Plasmids
Plasmid encoded His-Ubiquitin, Flag-WT-TRABID, Flag-C443S-TRABID, V5-WT-TRABID, 3xFlag-VPS34 were described in previous study (Liu et al., 2016; Yuan et al., 2014). Two TRABID fragments—3NZF and ankyrin repeat—were generated by PCR and cloned into pET32a vector. EGFP-ATG14L and EGFP-UVRAG were kindly provided by Dr. Guang-Chao Chen. For establishing stable overexpression clone, Flag-TRABID were subcloned into the lentivirus-based vector pLAS5W.Pneo.
Antibodies and Reagents
Mouse flag (M2; Sigma), goat TRABID (GeneTex), rabbit anti-GAPDH (GeneTex), rabbit anti-LC3B (Abcam), rabbit anti-LC3B (Cell Signaling), rabbit anti-VPS34 (PI3 Kinase Class III; Cell Signaling), rabbit anti-Beclin1 (Novus), rabbit anti-Ambra1 (Novus), rabbit anti-Flag (DDDDK, GeneTex), rabbit anti-β-actin (GeneTex), mouse anti-6xHis (Clontech), rabbit anti-V5 (Millipore), rabbit control IgG (Abcam), rabbit anti-VPS34 for IP (Echelon), rabbit anti-VPS34 (life technologies), rabbit VPS15 (PIK3R4; Novus), rabbit Beclin1 (Santa Cruz), EasyBlot anti-rabbit IgG (GeneTex), mouse anti-α-Tubulin (Millipore), mouse anti-GFP (Santa Cruz) antibodies were purchased from commercial sources. Rabbit anti-Rubicon and Rabbit anti-EGFR were kindly provided by Dr. Guang-Chao Chen. Coomassie Brilliant Blue R-250 and lysozyme was purchased from USB and BSA (Bovine serum albumin) was obtained from NEB. IPTG (Isopropyl β-D-thiogalactoside), DTT (1,4-Dithiothreitol), Bafilomycin A1 and cycloheximide were bought from Sigma-Aldrich, whereas recombinant human EGF protein was purchased from R & D systems.
Antibodies Production
Two TRABID fragments—3NZF and ankyrin repeats—were generated PCR and cloned into pET32a vector, which carries a 6xHis tag, followed by transformation into Rosetta competent cells. Cells were inoculated at 37℃ in LB containing 100 µM/ml of Ampicilin until OD600 reached 0.6, then induced with 0.5mM IPTG at 16℃ for 16-18 hours. For ankyrin repeats, recombinant proteins were purified under native condition.
Cells were pellet down and lysed with lysis buffer (100mM NaCl, 20mM Tris-HCl [pH=8.0], 1% NP40, adjust to pH=7.5) supplemented with protease inhibitor, 5mM DTT, 10mg/ml lysozyme. After six cycles of sonication (sonicate 20 sec, rest 20 sec as one cycle) and centrifugation, supernatants were transferred to 15 ml tube and incubated with Ni sepharose (GE Healthcare) at 4℃ for 2 hours. Beads were washed with wash buffer (300mM NaCl, 50mM Tris-HCl [pH=8.0], 20mM imidazole, adjust pH to 6.3) for three times and eluted with elution buffer (300mM NaCl, 50mM Tris-HCl [pH=8.0], 250mM imidazole, adjust pH to 7.5) at 4℃ for 30 min with agitation, which were repeated for two times. In the other hand, 3NZF were purified under denaturing condition. Cells were lysed with lysis buffer (300mM NaCl, 50mM Tris-HCl [pH=8.0], 1mM EDTA, adjust to pH=8.0) supplemented with protease inhibitor, 5mM DTT, 2%
Triton X-100, 10mg/ml lysozyme. Followed by sonication and centrifugation, insoluble pellet were washed with denaturing wash buffer (300mM NaCl, 50mM Tris-HCl [pH=8.0], 2M urea, 1mM EDTA, adjust pH to 6.3) supplemented with 1% Triton X-100 and 5mM DTT for three times then washed for another three times with wash buffer free from urea and Triton (300mM NaCl, 50mM Tris-HCl [pH=8.0], 1mM EDTA, adjust pH to 6.3). Insoluble proteins were extracted from the wash pellet with warm extraction buffer (300mM NaCl, 50mM Tris-HCl [pH=8.0], 6M guanidine hydrochloride, adjust to pH=8.0) supplemented with 20mM β-mercaptoethanol and
1mM PMSF rocking at 37℃ for 30min. Supernatants containing extracted proteins were collected after centrifugation and the denaturing binding buffer (300mM NaCl, 50mM Tris-HCl [pH=8.0], 2M urea, 20mM imidazole, adjust pH to 7.5) were added then subjected to incubation with Ni sepharose at 4℃. Beads were washed with denaturing wash buffer (300mM NaCl, 50mM Tris-HCl [pH=8.0], 20mM imidazole, adjust pH to 6.3) for three times and eluted with denaturing elution buffer (300mM NaCl, 50mM Tris-HCl [pH=8.0], 2M urea, 500mM imidazole, adjust pH to 7.5) at 4℃.
Purified antigens were provided to LTK BioLaboratories for immunization followed by 7 times of boosting. For antibody purification by affinity chromatography, soluble antigens were required for column packaging. To remove urea presenting in eluted antigens that were purified under denaturing conditions, eluted antigens were loaded into dialysis cassettes (ThermoFisher) and dialyzed in three different dialysis buffer with decreasing concentration of urea (1M urea and 1mM DTT in 1x PBS, 0.5M urea in 1x PBS, 1xPBS).
Cell Culture and Transient Transfection
293T, 293FT cells were cultured in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/
streptomycin (P/S). HeLa cells were maintained in Minimum Essential Medium (MEM) containing 10% FBS, 1% P/S and 1mM sodium pyruvate. For starvation, cells were cultured in Earle’s Balanced Salt Solution (EBSS; Sigma-Aldrich). Transient transfection of 293T was performed using calcium phosphate method. Cell medium was refreshed after 16-18 hours of incubation, and the cells were harvested 48 hours after transfection.
Lentivirus Production and Infection
Recombinant lentiviruses were packaged in 293FT cells by transiently transfecting 14 µg packing plasmid (pCMV△8.91), 2 µg envelope VSV-G plasmid (pMD.G) together with 14 µg Flag-TRABID cloned in pLAS5w.Pneo vector using calcium phosphate method. Cell medium was refreshed after 8-12 hours of incubation, and the supernatant containing packaged viruses were harvested after 42-48 hours of transfection then filtered by 0.45 µm pore-size syringe filter.
For infection, the supernatant were supplemented with 8 µg/ml polybrene and the infected cells were selected with 800 µg/ml neomycin. The constructs mentioned above were obtained from National RNAi Core Facility (NRC), Academia Sinica, Taiwan.
Western Blot
Cells were lysed by 1x RIPA buffer (150 mM NaCl, 20 mM Tris-HCl [pH=7.5], 1% NP40, 0.1%SDS, 1% sodium deoxycholate) with protease inhibitors (1mM PMSF, 1 µg/ml aprotinin and 10 µg/ml leupeptin). After sonication and centrifugation, supernatants were quantified by Bradford (Bio-Rad, Hercules, CA) and prepared with sample buffer (50mM Tris-HCl [pH=6.8], 2% SDS, 10% glycerol, 0.02% bromophenol blue, 2.5% β-mercaptoethanol) that is 1/4 volume of the sample. Protein samples were resolved by SDS-PAGE then transferred onto PVDF membranes (Millipore) that are activated by methanol beforehand. Membranes were blocked with 1-5% non-fat dry milk or 1% BSA in 1x TBST (Tris-buffered saline with 0.1% Tween-20) at room temperature for at least 20 min then incubated with indicated primary antibodies, which is diluted in blocking buffer, at 4℃ overnight. Next, membranes were washed 5 min for three times with 1 x TBST then incubated with indicated HRP-conjugated secondary antibody at room temperature for an hour. After washed 10 min for three times with 1x TBST and rinse with ddH2O, membrane were subjected to detection by immersing in
Western HRP substrates purchased from Millipore (LuminataTM Crescendo) or PerkinElmer, Inc. (Western Lightning® Plus-ECL)
Immunofluorescence
2.3x105 of HeLa cells stably expressing Flag-TRABID were seeded on glass coverslips, which was placed at the bottom on the 6-well plates, one day before the experiment. For serum starvation, cells were washed with 1x PBS (phosphate-buffered saline) for two times and EBSS for one time then cultured with EBSS for the indicated time. Cells were washed with 1x PBS for two times and fixed with 4% formaldehyde for 20-25min on ice. After washed with 1xPBS for three times, cells were permeabilized at room temperature with -20℃ methanol for 10 min and washed another three times with 1x PBS. Cells were then blocked with blocking reagents (1% BSA, 10% goat serum in 1xPBS) at 4℃ for 1hr, and incubated with anti-LC3B antibody diluted with blocking reagents at 4℃ overnight . After washed with 1xPBS for three times, cells were rinsed with blocking reagents and subjected to fluorescent dye-conjugated secondary antibodies (life technologies) and DAPI (Sigma-Aldrich) at room temperature for 1 hour. Stained cells were washed with 1x PBS for three times, mounted onto microscope slides with fluorescence mounting medium (Dako), then examined by confocal microscope (Zeiss LSM510) with 63x oil objective lens. A total of approximately 100 cells were analyzed in each group and the average puncta/cell was quantified.
Immunoprecipitation
For Fig. 4 and Fig. 8A, cells were lysed with 1x RIPA supplemented with protease inhibitors and phosphatase inhibitors (1mM Na3VO4, 2mM NaF, 200µM NaPPi). Followed the above-mentioned protocols, cell lysates containing equal amount
of proteins were incubated at 4℃ for 2 hours with anti-Flag agarose beads (M2; Sigma-Aldrich) or anti-V5 agarose beads (Sigma-Sigma-Aldrich) respectively. After washed three times with lysis buffer and prepared with sample buffer, immunoprecipitants were subjected to Western blot analysis. While in Fig. 8B, same protocol was applied except replacing the lysis buffer with 1x NP40 buffer (150mM NaCl, 50mM Tris-HCl [pH=7.5], 1% NP40) and the beads with GFP-Trap®_A (chromotek).
For endogenous co-immunoprecipitation in Fig. 6, the other kind of lysis buffer, named “Lysis Buffer-C” (100mM NaCl, 10mM Tris-HCl [pH=7.5], 2mM EDTA, 1%
NP40), supplemented with protease and phosphatase inhibitors were used. Equal amount of cell lysates were first pre-cleared at 4℃ for 40-60min with protein A sepharose (GE Healthcare). Then, the supernatants were incubated with 5 µg of control IgG or anti-VPS34 antibody at 4℃ overnight followed by 2 hours of incubation with protein A. Immunoprecipitants were subjected to Western blot analysis followed the standard protocols, except for replacing the secondary antibody with EasyBlot anti-rabbit IgG.
In vitro Deubiquitination Assay
For substrate purification, 293T cells were transfected with His-Ubiquitin together with 3xFlag-VPS34. After 48 hours of transfection, cells were lysed with 2x RIPA and incubated with anti-Flag agarose beads for 2 hours. Purified ubiquitinated substrates were washed with 2x RIPA for five times and 2x elution buffer (300mM NaCl, 20mM Tris-HCl [pH=7.5]) for one time. Ubiquitinated substrates were eluted with 2x elution buffer containing 150 ng/ml of 3xFlag peptides by agitating at 4℃ for 30 min, which were repeated for two times. Concentration of eluted proteins were quantified by SDS-PAGE relative to BSA. Purified substrates could be stored at -80℃.
For enzyme preparation, 293T cells were transfected with V5-WT-TRABID. After 48 hours of transfection, cells were lysed with 1x RIPA and incubated with anti-V5 agarose beads for 2 hours. Purified enzymes were washed with 1x RIPA for three times and 2x reaction buffer (300mM NaCl, 20mM Tris-HCl [pH=7.5], 0.4% NP40, 0.2% Triton X-100) for one time. After draining the washing reagents, same volume of 2x reaction buffer containing 20 mM DTT was added to the beads for resuspension and incubated at 23℃ for 10 min to restore the full activity of enzymes. Purified TRABID proteins were incubated with 500-1000 ng of substrates at 37℃ for 4 hours with agitation. After the reaction was terminated by preparing with sample buffer, samples were resolved by Bis-Tris Gel using 1x MOPs running buffer (50 mM MOPs, 50mM Bis-Tris-HCl, 1mM EDTA, 0.1% SDS, 5mM sodium bisulfate) then followed by standard protocol of Western blot analysis.
Cycloheximide-Chase Assay
293T cells that stably expressing two different TRABID shRNAs were seeded on 6-well plates a day before the experiment, whereas 293T cells that transiently transfected with Flag-TRABID were subjected to cycloheximide treatment 36-42 hours after transfection. Cells were incubated with medium containing 100 µg/ml of cycloheximide for indicated time and the lysates were subjected to Western blot analysis. VPS34 protein level was first quantified relative to β-actin using image J and then normalized to the amount of untreated cells.
Mass PI3P ELISA
HeLa cells stably expressing empty vector or Flag-TRABID were seeded on 10 cm plates a day before the experiment. To extract lipid from cells, cells were scraped with ice cold 0.5M TCA followed by centrifugation. Pellet were collected and wash two
times with 5% TCA containing 1mM EDTA. Pellets were washed another two times with MeOH:CHCl3(2:1) to eliminate neutral lipids. Acidic lipids were extracted by applying MeOH:CHCl3:12M HCl (80:40:1). Supernatants were subjected to phase spit step through adding CHCl3 containing 0.1N HCl and the organic phase (lower phase) were collected to eppendorfs for 30 min of vacuum drying by SpeedVac. Extracted lipids were then assayed by using PI3P Mass ELISA kit (Echelon, K-3300). Extracted lipids were rehydrated and the samples and PI3P standards were transferred to incubation plate for following incubation on shaker with PI3P detector at room temperature for an hour. Mixture of each well was transferred to the corresponding well of detection plate and incubated for another hour. After washing three times with PBS-T, secondary detectors were added to each well and incubate at room temperature for an hour. Followed by three times of washing, TMB solution were added and incubated for 30 min in dark to develop the color. The reaction was stopped by adding 1N H2SO4, and the plate were presented to Beckmen Paradigm for absorbance measurement.
EGFR Degradation Assay
HeLa cells stably expressing empty vector or Flag-TRABID and 293T cells stably expressing control or TRABID shRNA were seeded on 6-well plates a day before the experiment. When the confluency reached 80% the next day, cells were washed two times with PBS and serum-starved overnight in medium containing only MEM or DMEM. On the third day, cells were stimulated with medium (MEM or DMEM supplemented with 20mM HEPES, 0.2% BSA) containing 100 ng/mL EGF for the indicated time. EGFR protein level was quantified relative to β-actin using image J and normalized to the amount of untreated cells.