Protease inhibitor and phosphatase inhibitor

Cells were collected and lysed with RIPA buffer (Beyotime) supplemented with protease inhibitor and phosphatase inhibitor (Bimake). Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). The membranes were incubated overnight at 4 °C with the primary antibodies listed in Table S1, and then horseradish peroxidase-coupled secondary antibodies were performed at 37 °C for 1 h. The antigen–antibody reaction was observed by enhanced chemiluminescence.

Cell lysates were prepared using RIPA buffer (Thermo Fisher Scientific, Shanghai, China) or IP Lysis buffer (Thermo Fisher Scientific, Shanghai, China) supplemented with protease inhibitor and phosphatase inhibitor (Bimake, Shanghai, China). Equal amounts of cell lysates were fractionated by SDS-PAGE and then blotted onto nitrocellulose membranes. Membranes were blocked for 1 h at room temperature (RT) with 5% nonfat milk and then probed with specific primary antibodies for 2 h at RT. After being washed with TBST (10 min for each wash), the membranes were incubated with either horseradish peroxidase (HRP)-conjugated goat anti-rabbit or goat anti-mouse antibody (Proteintech, Wuhan, China, 1:6000;) for 1 h at RT. After the membranes were washed three times, signals were raised using an ECL kit (Thermo fisher, Shanghai, China) and detected with a Tanon-5200 automatic chemiluminescence image analysis system. The primary antibodies used were as follows: mouse anti-HA (Sigma, Shanghai, China, 1:6000), rabbit anti-Flag (Sigma, Shanghai, China, 1:6000), mouse anti-β-actin produced (Sigma, Shanghai, China, 1:6000), N polyclonal antibody produced in mouse (1:500; reserved in laboratory), mouse anti-PCSK9 (Detai Biotechnology, Nanjing, China, 1:1000), and mouse anti-MYC (Cell Signal Technology, Shanghai, China, 1:1000;).

The co‐IP assays were performed as previously described.^[40 (link)
^] Briefly, 3 × 10⁶ cells were lysed with NP‐40 lysis buffer (0.5%, pH 7.4) containing 20 mm Tris, 100 mm NaCl, and 0.5 mm EDTA supplemented with 1% protease inhibitor and phosphatase inhibitor (Bimake, Cat # B14001, B15001) for 30 min at 4 °C, followed by incubation with 1 µg primary antibodies at 4 °C overnight. Then, cell lysate was incubated with protein G plus protein A agarose beads (Merck, Cat # IP10‐10MLCN) for 2 h at 4 °C. Next, the beads were washed three times using NP‐40 wash buffer with similar constituents as in the NP‐40 lysis buffer except for NaCl (150 mm). The beads were then boiled with SDS‐PAGE loading buffer for 10 min and subjected to immunoblotting analysis.

Radioimmunoprecipitation assay (RIPA) buffer (Cell Signaling Technology, US) supplemented with protease inhibitor and phosphatase inhibitor (Selleck, US) was used to collect cellular proteins. Then, the protein concentration was evaluated using the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, US). SDS-polyacrylamide gel electrophoresis assay and transfer to PVDF membranes were performed followed by incubation for an hour in blocking buffer containing 5% skimmed milk. Next, primary antibodies were incubated overnight at 4℃. The next day, after incubation with HRP-conjugated rabbit or mouse secondary antibody (Santa Cruz Biotechnology, US) for 2 hours at room temperature, chemiluminescence reagent was used, and blots were visualized using a chemiluminescence imager (Bio-Rad, US) and analyzed using Image Lab Software (Bio-Rad, US) or physical film in the darkroom. The antibodies used were NPTX2 (Cat. No. 10889-1-AP, Proteintech, the US) and GAPDH (Cat. No. 10494-1-AP, Proteintech, the US).

Total protein was extracted from cell lines lysed with RIPA buffer (Beyotime, Shanghai, China) in the presence of a protease inhibitor and phosphatase inhibitor (Selleck, Shanghai, China). The concentration of protein was determined by BCA kit (Beyotime). Protein was resolved by SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Burlington, MA). The membranes were blocked with 5% non-fat milk in TBST for 2 h at room temperature and then incubated with primary antibodies overnight at 4°C. The primary antibodies include rabbit anti-TIPE2 (ProteinTech), anti-pERK, anti-ERK, anti-pAKT, anti-AKT, anti-Bax (Cell Signaling Technology), anti-GAPDH (ProteinTech), anti-pTGFBR1 and anti-TGFBR1 (Affinity, Cincinnati, OH). After washing, membranes were incubated with HRP-conjugated secondary antibody (goat anti-rabbit IgG or goat anti-mouse IgG, Cell Signaling Technology) for 1 h at room temperature. After washing, the protein bands were visualized with enhanced chemiluminescence (ECL) detection kit (Beyotime).

Cells were harvested in RIPA Lysis Buffer (DingGuo) with a protease inhibitor and phosphatase inhibitors (Selleck), and protein concentration was determined using a BCA assay kit (Beyotime). Protein samples (30 μg) were subjected to 8%‐12% SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 5% non‐fat milk or 5% bovine serum albumin (BSA) for one hour at room temperature and then incubated with following primary and secondary antibodies: USP10 (1:1000, CST), USP13 (1:1000, Proteintech Group), PARP (1:1000, CST), BAX (1:1000, CST), BCL2 (1:1000, CST), CDC2 (1:1000, Proteintech Group), CyclinB1 (1:1000, Santa), γ‐H2AX (1:1000, CST), p‐ATM (1:1000, CST), p‐ATR (1:1000, CST), ATM (1:1000, Proteintech Group), ATR (1:1000, Proteintech Group) or GAPDH (1:3000, Proteintech Group). The blots were detected and analysed using a gel image analysis system (LI‐COR).