Anti gapdh

RIPA buffer containing protease inhibitors were used to extract total proteins from tissues and cultured cells and the concentrations were measured by bicinchoninic acid (BCA) solution (Beyotime, Nantong, China). The Western Blot was performed under the standard program and the primary antibodies, anti-GAPDH, as well as the secondary antibodies, including anti-rabbit HRP-linked and anti-mouse HRP-linked were from Beyotime (Nantong, China). Besides, anti-AKT3 was from lifesbiology(Wuxi, China) .

Nuclear and cytoplasmic extracts were prepared using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Institute of Biotechnology, Jiangsu, China) as described previously.^{11 (link)} Total proteins were extracted using RIPA lysis buffer. Fifty micrograms of cell lysates were electrophoresed with 10% SDS-PAGE. The specific primary antibodies used against each protein in the immunoblotting were as follows: anti-Drosha (1:1000; Abcam), anti-PCNA (1:800; Abcam), anti-LAMC2 (1:1000; Millipore), anti-CD82 (1:1000; CST); the antibodies anti-EGFR, anti-p-EGFR, anti-ERK1/2, anti-p-ERK1/2, anti-MMP7 and anti-GAPDH (1:1000; all from Beyotime); anti-β-tubulin (1:500; Santa Cruz Biotechnology, Santa Cruz, TX, USA) and anti-β-actin (1:1000; Boster, Wuhan, China). The appropriate horseradish peroxidase-conjugated secondary antibodies were subsequently applied. The proteins were visualized by the enhanced chemiluminescence system (Amersham, Pharmacia Biotech, Freiburg, Germany).

Proteins in cells and tissues were extracted with RIPA lysis buffer (Thermo Fisher). Serum proteins were extracted with Serum Protein Extraction Kit (Qcheng Bio, China). Western blot assays were performed according to details previously reported [23 (link)]. the immuno-complexes were detected with ECL Western Blotting Substrate (Thermo Fisher), visualized with BIO-RAD (BIO-RAD Gel Doc XR+, USA). The following antibodies were used (11000): anti-β-actin (Beyotime, AF0003); anti-α-tubulin (Beyotime, AF0001); anti-GAPDH (Beyotime, AF0006); anti-HSP90 (Proteintech, 60,318–1-Ig); anti-HUR (Proteintech, 11,910–1-AP); anti-EIF2S1 (Proteintech, 11,170–1-AP); anti-VEGF (Proteintech, 19,003–1-AP); anti-Calnexin (Abcam, ab92573); anti-CD63 (Abcam, ab134045); anti-TSG101 (Abcam, ab125011); anti-CD81 (Proteintech, 66,866–1-Ig); anti-STUB1 (Proteintech, 55,430–1-AP).
IHC, IF and IP was performed as previously described [24 (link)]. IHC was performed with antibodies against HUR and VEGF (Proteintech, 1:200). IF was performed with antibody against CD31 (Proteintech, 11,265–1-AP, 1:200). The images were scanned by Pannoramic SCAN (3DHistech, Hungary). IP was performed with anti-HSP90 antibody (Proteintech, 1:200) and appropriate control IgG (Merck Millipore), and the immunoprecipitate was then collected by centrifugation and analysed by SDS-PAGE.

Proteins were isolated from transfected cells by RIPA lysis buffer (Beyotime, Nantong, China) containing 0.5% PMSF. The total proteins (50 μg per protein) were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were incubated for 1 h in blocking solution (Beyotime) at room temperature and then immunoblotted overnight at 4°C with the following primary antibodies: anti-MDMX (1:1,000, Proteintech Group, Rosemont, IL, USA), anti-P53 (1:1,000, Proteintech Group, Rosemont, IL, USA), anti-P21 (1:1,000, Proteintech Group, Rosemont, IL, USA) and anti-GAPDH (1:1,000, Beyotime). And enhanced chemiluminescence reagent kit (Millipore, Billerica, MA, USA) was utilized for exposure after the blot incubated with secondary antibody (Beyotime) for 1 h.

Proteins were isolated via NP40 lysate, electrophoresed in 10% SDS-polyacrylamide and transferred to polyvinylidene fluoride membrane (Millipore, USA). It was treated with anti-FOXO1 (1:500, ab52857, Abcam, UK) or anti-GAPDH (1:1000, Beyotime, China) at 4 °C for a night. The membranes were then incubated in secondary antibody for an hour at 25 °C. After washing, we detected the signal using the ECL kit (P0018, Beyotime, China).

Celecoxib, cisplatin, 5-fluorouracil (5-FU) and prostaglandin E₂ (PGE₂) were purchased from Sigma-Aldrich. Each compound was prepared as 1 mM stock solution in dimethylsulfoxide (DMSO) for dilution into various concentrations. The following mouse or rabbit monoclonal primary antibodies were used: anti-E-cadherin (Abcam), anti-MMP-2 (Abcam), anti-Vementin (Abcam), anti-c-MYC (Abcam), anti-Axin-2 (Abcam), anti-Cyclin-D1 (Abcam), anti- β-catenin (Cell Signaling Technology), anti-Survivin (Cell Signaling Technology), anti-SOX-2 (Cell Signaling Technology), anti-p-GSK-3β (Cell Signaling Technology), anti-GAPDH (Beyotime Biotechnology) and anti-Tubulin (Beyotime Biotechnology). HRP conjugated goat anti-mouse or anti-rabbit secondary antibodies were purchased from Beyotime Biotechnology.

RIPA Lysis Buffer (Beyotime, China) containing PMSF (Beyotime, China) and PhosSTOP (Sigma-Aldrich, USA) was used to lyse the cells with indicated treatment. After quantification, the protein samples were separated by SDA-PAGE gel (Beyotime, China). Primary antibodies (Anti-RIPK1, BD Biosciences, USA; Anti-RIPK3, Abcam, USA; p-RIPK3, Abcam, USA; Anti-MLKL, Sigma-Aldrich, USA; Anti-p-MLKL, Sigma-Aldrich, USA; Anti-GAPDH, Beyotime, China) were used at 4 °C overnight. The protein bands were detected by ECL Kit (BOSTER, China).