Anti gapdh 10494 1 ap

Antibodies utilized in this study are as follows: anti-α-SMA (GTX629702) from GeneTex; anti-USP9X (ab19879), anti-NRP1 (ab81321), Anti-Myc tag (ab9106), anti-Flag-Tag (ab205606), and anti-GST (ab92) from Abcam; anti-collagen I (91144S), anti-rabbit IgG (3900), anti-mouse IgG (5415), from Cell Signaling Technology; anti-GAPDH (10494-1-AP) from Proteintech.

The protein expressions of Occludin and β defensin in the colon wall were determined by the standard Western blot method with the GAPDH protein as a loading control. The anti-Occludin (#13409-1-AP) and anti-GAPDH (10494-1-AP) primary antibodies were obtained from Proteintech (Wuhan, Hubei, China). The anti-β defensin (#bs-1296R) primary antibody was obtained from Bioss (Bioss Biotech, Beijing, China). The horseradish peroxidase-conjugated secondary antibody was obtained from Cell Signaling Technology Co.

Total protein was extracted using RIPA buffer (Beyotime), supplemented with protease inhibitor. Protein concentrations were validated by the BCA assay (Sigma–Aldrich, Cambridge, MA, USA). Proteins were separated by SDS–PAGE gels and were then transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 5% fat-free milk in TBS-T for 2 h at room temperature. Next, primary antibodies were incubated overnight at 4°C. The following day, secondary antibodies were incubated for 1.5 h at room temperature. The primary antibodies were anti-LC3 (14600-1-AP, Proteintech, USA), anti-p62 (ab207305, Abcam, USA), anti-EZH2 (#5246, Cell Signaling Technology, USA), anti-ATG5 (10181-2-AP, Proteintech, USA), and anti-GAPDH (10494-1-AP, Proteintech, USA).

The concentrations of total protein extracted from the cells were quantified using a BCA assay (MCE, China). After separation using 10% SDS-PAGE, the proteins were transferred to PVDF membranes (Millipore, United States) and then incubated with the corresponding primary antibody and horseradish peroxidase-conjugated secondary antibody according to the manufacturer's instructions. Protein visualization was performed using an ECL kit (HYK1005, MCE, China). Protein expression was quantified using ImageJ software (NIH, United States). Anti-LAST1 (17049-1-AP), anti-Bcl-2 (12789-1-AP), anti-Bax (1 : 2000, 50599-2-Ig), anti-GAPDH (10494-1-AP), and anti-β-actin (66099-1-Ig) were purchased from Proteintech, China. Anti-YAP1 (#14074) and anti-phospho-YAP1 (#13008) were purchased from CST, United States. The secondary antibodies (1 : 2000) were purchased from Servicebio, China. The antibodies mentioned above were diluted at a ratio of 1 : 1000 unless stated otherwise.

Quantitative RT-PCR and WB were carried out as previously described [20 (link)]. RT-PCR reagents were purchased from Accurate Biology (Changsha, China), which included the SYBR Green Premix Pro Taq HS qPCR Kit, AG RNAex Pro Reagent, and Evo M-MLV RT Premix Kit. Supplementary Table 1 shows the primer sequences. Beyotime Biotechnology (Shanghai, China) provided WB reagents, such as BeyoECL Plus and RIPA buffer Kit. Proteintech (Wuhan, China) supplied anti-OGT (11576-2-AP) and anti-GAPDH (10494-1-AP) antibodies.

GL261 cells were lysed in RIPA buffer (P0013E, Beyotime) containing protease inhibitor (BL612A, Biosharp) and phosphatase inhibitor (AR1183, Boster) cocktail. Protein concentration was measured using a BCA assay (P0010S, Beyotime). Anti-ASB3 (AP16752a, Abcepta) was used as the primary antibody, anti-GAPDH (10494-1-AP, Proteintech) was used as the control, and IRDye^® 800CW Goat anti-Rabbit IgG (D10629-12, LI-COR) was used as the secondary antibody. Western blotting was detected automatically using the Odyssey Infrared Imaging System (LI-COR).

Protein was extracted from HUVECs using RIPA Lysis Buffer (Millipore, USA), and protein concentration was measured using the BCA Protein Quantitative Kit (Beyotime, China). The proteins were separated by SDS-PAGE and then transferred to a PVDF membrane. The following primary antibodies were used: rabbit monoclonal anti-HIF-1α (ab179483; Abcam, UK), rabbit polyclonal anti-VEGF-A (ab46154; Abcam), rabbit polyclonal anti-EIF4E3 (17282-1-AP; Proteintech, USA), rabbit polyclonal anti-EIF4E1 (17282-1-AP; Cell Signaling Technology, USA), and rabbit polyclonal anti-GAPDH (10494-1-AP; Proteintech). After incubation with the primary antibodies, the membrane was incubated with horseradish peroxidase-labeled goat-anti-rabbit IgG (ZB-2301, ZSGB-Bio, China). The immunoproteins were detected using the Hypersensitivity ECL Luminescence Kit (Beyotime), and band densities were quantified using Quantity One v4.6.2 (Bio-Rad).