Gapdh antibody

Specific pathogen-free (SPF) grade 4-week-old (70-80 g) male SD rats were purchased from Hunan Changsha Tianqin Biotechnology Co., Ltd. Common feed, high-fat feed, and streptozotocin (STZ) were all obtained from Beijing Boai Port Biotechnology Co., Ltd. Cx43 antibody (#AF0137), mTOR antibody (#AF6308), AKT antibody (#AF6261), GAPDH antibody (#T0004), β-actin antibody (#AF7018), LC3 antibody (#AF5402), p62 antibody (#AF5384), goat anti-rabbit IgG (#S0001), and goat anti-mouse IgG (#S0002) were all purchased from Affinity Biosciences (Affinity Biosciences Milwaukee, WI 53224, America). Dapagliflozin was provided by AstraZeneca Pharmaceutical Co., Ltd.

GI-LI-N and SK-N-SH cells were harvested and lysed with RIPA cell lysis buffer (Thermo Fisher Scientific) with protease inhibitors. The protein concentration was measured by bicinchoninic acid method (Thermo Fisher Scientific). Then the extracted protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto the polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA) on ice. Next, after being blocked for 1 h with 5% non-fat milk at room temperature, the PVDF membranes were incubated with primary antibodies overnight at 4°C. MMP-2 antibody (1:2,000), E-cadherin antibody (1:2,000), N-cadherin antibody (1:1,000), vimentin antibody (1:1,000), phosphatidylinositol 3-kinase (PI3K) (#4,257, 1:1,000), phosphorylated (p)-PI3K (#17,366, 1:1,000), serine/threonine kinase 1 (AKT) (#9,272, 1:1,000), p-AKT (#9,611, 1:1,000), and GAPDH antibody (1:1,000) were purchased from Affinity Biosciences (Changzhou, China). Moreover, membranes were incubated with the corresponding HRP-conjugated second antibodies HRP Goat Anti-Rabbit IgG (H+L) (1: 5000, ABclonal, Wuhan, china) or HRP Goat Anti-Mouse IgG (H+L) (1: 5000, ABclonal), and protein signals were visualized with enhanced chemiluminescence (Millipore).

Total proteins were extracted using a protein extraction kit (Beyotime Institute of Biotechnology Co., Shanghai, China) according to the manufacturer’s instructions. Protein content was quantified using the BCA protein assay reagent (Beyotime Institute of Biotechnology Co., Shanghai, China), and protein was diluted to the same concentration. Protein (25 μg per lane) was separated using sodium dodecyl sulfate-polyacrylamide gels electrophoresis, and was electro-transferred onto polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were then incubated with either TNF-α and TNFR1 antibodies (Wanleibio Biological Technology Co., Ltd., Shenyang, China), p-IκB α and IκB α antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA), or NF-κB p65 and p-NF-κB p65 antibodies (Abcam, Cambridge, UK). A GAPDH antibody (Affinity Bioscience (China) Co., Hong Kong, China) was used as the internal control. Subsequently the PVDF membranes were incubated with an appropriate secondary antibody (Affinity Bioscience (China) Co., Hong Kong, China). Finally, protein bands were observed using an enhanced chemiluminescent reagent (Biosharp Life Sciences, Hefei, China). Protein grey intensity was quantified by the Fiji/ImageJ-win 64 program normalized GAPDH levels. Each western blot was performed a total of three times.

Human monocytes (1.2 × 10⁶) obtained using our routine procedure were lysed by EzRIPA Lysis buffer (Atto, Tokyo, Japan) and protein concentration was determined using Micro BCA™ Protein Assay kit (Thermo Fisher, Tokyo, Japan). Protein lysate was kept in ice until use for pull‐down assay. Thereafter, 100 μL of His60 Ni Magnetic Beads (Takara, Osaka, Japan) was washed by PBS, then was incubated with 10 μg His‐tagged TYMP (ATGEN, Korea) using slow‐speed rotator (AS ONE, Osaka, Japan) for 2 h. The TYMP‐bound beads were next washed by PBST and incubated with 1 mL lysate protein containing 250 μg protein concentration for 2 h. Negative controls included lysate and beads without recombinant TYMP. Protein‐bound‐beads were washed 3 times by PBST and finally subjected to SDS‐PAGE and Western blot analysis. Bound proteins were first tested by Western blot analysis with antibody to irreverent target (GAPDH antibody; Affinity Biosciences) to validate our assay. No reaction was detected with GAPDH antibody (data not shown).