Mg132

Human non-transformed bronchial epithelial cell line Beas2B, and LC cell lines H661, SPC-A1, SK-MES-1, H1299, H1373, H292 and A549 were all from the Chinese Infrastructure of Cell Line Resources. A549, H661, SPC-A1 and H292 cells were cultured in RPMI 1640 medium containing 10% FBS, and Beas2B, H1299, H1373 and SK-MES-1 cells were cultured in DMEM medium containing 10% FBS. All cell lines were maintained in an incubator containing 5% CO₂ at 37°C. To induce EMT, H1299 and A549 cells were starved in medium containing 0.1% FBS for 16 h before treatment with 5 ng/mL TGF-β1 (ProSpec, Israel) for indicated hours. CHX (100 μg/mL; Sigma, USA) was used to treat H1299 cells for 0, 0.5, 1, 2, 3 h to interdict new β-catenin synthesis. To inhibit β-catenin degradation, H1299 cells were treated with proteasome inhibitor MG132 or GSK3β inhibitor SB415286 (25μM; MedChem Express, China) and harvested at 4 h for MG132 and 12 h or 24 h for SB415286.

Huh7 cells were maintained in mediums containing 0, 5, 10, 20, 25 ng/ml TPL for 24 h or 48 h before being harvested for determination of c-FLIP protein and mRNA. To confirm the effects of TPL on c-FLIP protein expression were not cell line specific, Hep3B cells were also incubated with TPL for 24 h followed by analysis of c-FLIP levels by Western blot. Besides, Huh7 cells were incubated with medium containing 20 ng/ml TPL for 0, 6, 12, 22, 32 h and then collected for evaluation of c-FLIP protein levels.
Moreover, Huh7 cells were untreated or pre-treated with proteasome inhibitor Lactacystin (LC) (APExBIO Technology LLC, Houston) or MG132 (MedChemExpress, Shanghai) for 2 h before the addition of TPL, and 4 h, 8 h, 12 h (for cells pre-treated with LC) or 2 h, 4 h, 6 h (for cells pre-treated with MG132) after TPL was added in, cells were harvested for analysis of FLIP_S levels.

HBE41o- and CFBE41o- expressing WT and F508del CFTR, respectively, were obtained from Dr. J Clancy (University of Alabama). Cells were cultured in α-MEM containing 10% fetal bovine serum (FBS) and 2 mM l-glutamine. HBE41o- and CFBE41o- containing YFP-H148Q were engineered and cultured as previously described^{168 (link)}. siRNA-mediated silencing. HBE41o- and CFBE41o- cells were plated in 12-well dishes and grown to 60–70% confluence. COPII subunit silencing was performed using 50 nm of validated siRNA for the indicated COPII subunit as per manufacturer’s directions. Cells were cultured for 5 h in the serum-free opti-MEM containing transfection complexes and subsequently washed and cultured for an addition of 48 h in the presence of growth medium prior to processing for immunoblot analysis. Treatment. Where indicated, cells were treated with MG132 (10 µM), chloroquine (50 µM) in complete growth media and incubated at 37 °C, 5% CO2 for 24 h. MG132 and chloroquine were procured from MedChemExpress.

Cells were transfected with indicated plasmids. Forty-eight hours after transfection, the cells were treated with 20 µM MG132 (MedChemExpress, USA) for 4 h before harvesting. Cell extracts were lysed in NP-40 Lysis Buffer supplemented with protease inhibitor, and the lysates were incubated with GPX2 or HA tag antibody at 4°C overnight. Immune complexes were collected by adding 40 µL of Protein A/G PLUS-Agarose (sc-2003, SantaCruz Biotechnology, USA) for 6 h at 4°C. Immunoprecipitates were collected by centrifugation at 2500 rpm for 5 min at 4°C and analysed by western blot.

For regents, CHX (Sigma, St Louis, MO, USA), MG132 (MedChemExpress, Monmouth Junction, NJ, USA), 3-MA (MedChemExpress), CHQ (MedChemExpress), Bort (MedChemExpress), and IFNα (Sigma) were used to treat cells. For plasmids, ISG15 and 6PGL expression plasmids were bought from Origene (Beijing, China). LentiCRISPR v2 based constructs were used for knockout ISG15, UbCH8, HERC5, 6PGL, SMADs, TEADs. pGL4.21 vector was used to construct a 6PGL promoter-luciferase vector. YAP^K280R-HA, YAP^K321R-HA, YAP^K497R-HA, 6PGL^Mut-P1, 6PGL^Mut-P2, 6PGL^Mut-P3, and 6PGL^Mut-P1+P2+P3 mutant plasmids were constructed using overlapping PCR. YAP, ATG5, PSMB5, and βTrCP knockout constructs, YAP^WT-HA, YAP^FLAG, SMAD2, TEAD4, RUNX2, TFCP2, P73, pUAS-Luc/TEAD-Gal4 plasmids were acquired from previous studies [15 (link), 31 (link), 43 (link), 49 (link), 50 (link)]. The primers are listed in Supplementary Table 1.

The 293T cells were transfected with hemagglutinin (HA)-ubiquitin, Myc-WWP1, and either Flag-KLF2 or Flag-KLF2-MUT, followed by treatment with 10 μM MG132 for 4 h, and harvested. Cells were lysed in 100 μL of regular lysis buffer. The cell lysates were denatured at 95 °C for 5 min with the presence of 1% SDS, followed by overnight culture in anti-Flag antibody and protein G-agarose (Sigma-Aldrich, www.sigmaaldrich.com) overnight at 4 °C. Western blot analysis with an anti-HA antibody was conducted to analyze the immunoprecipitates.
BMSCs were first treated with 10 μM MG132 (MedChemExpress, Shanghai, China) for 4 h. Cell lysates were then cultured with anti-KLF2 antibody and protein G agarose overnight at 4 °C. The endogenous ubiquitination of KLF2 in the immunoprecipitates was assessed by Western blot analysis with the use of an anti-ubiquitin antibody.

Co‐immunoprecipitation and ubiquitination assays were carried out as described previously.²³ (link) Briefly, the cell lysates were blocked with 40 μl Protein A + G agarose beads (Beyotime Biosciences) at 4°C for 2 hours and immunoprecipitated with 4‐10 μg mouse primary anti‐p53 (#60283‐2‐Ig, Proteintech), mouse primary anti‐FLAG (#HT201‐01, TransGen Biotech) or rabbit primary anti‐p53 (#10442‐1‐AP, Proteintech) antibodies overnight at 4°C. The immune complexes were captured by 30 μl Protein A + G agarose beads and visualized via Western blotting.
For the ubiquitination assay, A549 cells were transfected with specified siRNA or plasmids for 48 hours and then treated with MG132 (MedChemExpress, Monmouth Junction) for 24 hours before they were collected and lysed.
After centrifuging, the supernatant was immunoprecipitated using an anti‐p53 (1:1000, #10442‐1‐AP/#60283‐2‐Ig, Proteintech) antibody overnight at 4°C. Samples were then incubated with Protein A + G agarose (Beyotime Biosciences) and washed there times in NP‐40 lysis buffer. Agarose and 20 μg of the protein immunocomplex were immunoblotted using an anti‐HA antibody (1:1000, #51064‐2‐AP, Proteintech).