Anti gapdh antibody

Protein samples were obtained, and Western blot analysis was performed according to the methods as previously described.⁴³ The antibodies used in the experiment are as follows: rabbit antibodies, such as anti‐p‐JNK (1:1000), anti‐JNK (1:1000), anti‐MKK4 (1:1000) and anti‐cbl‐b (1:1000), were purchased from ProteinTech Group, Inc; anti‐GAPDH antibody (1:30 000) was purchased from Zsgb Bio; anti‐NR4A1 antibody (1:1000) were purchased from Affinity Biosciences and Abcam; and mouse anti‐beta actin (1:5000) and rabbit anti‐α‐Tubulin (1:5000) were purchased from Bioworld Technology.

Twenty micrograms of the extracted villus protein sample was separated by SDS‒PAGE, transferred to a polyvinylidene fluoride (PVDF) membrane and sealed with 5% defatted milk powder for 1 h. The membrane was incubated with diluted primary antibodies overnight at 4 °C. The membrane was incubated with secondary antibodies for 1 h. The horseradish peroxidase (HRP) signal was detected using hypersensitive enhanced chemical luminescence (ECL) chemical reagent. The target protein bands were analyzed in ImageJ.
Human HSP90AB1 antibody (Boster, Wuhan, China, no. BM4191, 1:2000 dilution), human HSPD1 antibody (Boster, Wuhan, China, no. M01280-3, 1:2000 dilution) and human HSPA13 antibody (Proteintech, Wuhan, China, no. 12667–2-AP, 1:2000 dilution) were used as the primary antibodies for Western blot analysis. The membranes were also incubated with anti-GAPDH antibody (ZSGB-Bio, no. TA-08, 1:10,000 dilution) to verify equal protein loading.

Cells were purchased from the National Infrastructure of Cell Line Resource (Beijing, China). Papillary thyroid cancer cell lines TPC-1 and BCPAP were cultured for 48 h in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Cleveland, TN, USA), with 10% fetal bovine serum (FBS) (Gibco, Cleveland, TN) and 1% penicillin/streptomycin in a 37 °C/5% CO₂ incubator. Cell transfection was performed with Lipofectamine 2000 (Thermo Fisher-Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions. Small interfering RNA (siRNA-ANGPTL2: sc-72351) duplexes were purchased from Santa Cruz (Dallas, TX, USA). The ANGPTL2 siRNA was a pool of 3 target-specific 19–25 nt siRNAs designed to knock down ANGPTL2 gene expression. Full-length cDNA encoding human ANGPTL2 were cloned into the vector GV366 plasmid (Shanghai Genechem Co., Ltd) with a HA-tag. Cultured cells were harvested 48 h after transfection in sodium dodecyl sulfate (SDS) sample buffer and were analyzed by western blot to investigate the ANGPTL2 expression change. ANGPTL2 antibody (ab199133) was purchased from Abcam. Polyclonal rabbit anti-HA (#561) antibody was purchased from MBL (Nagoya, Japan). Anti-GAPDH antibody was obtained from ZSGB-BIO (Beijing, China). Otherwise, cells were collected at required time point for different treatment base on the requirement of different analysis methods.

Total proteins were obtained from tissues and cells lysed in RIPA buffer (KeyGen Biotech, Nanjing, China) supplemented with a protease inhibitor cocktail (Amresco, Solon, Ohio) and phosphatase inhibitor (KeyGen Biotech). The protein content was quantified using a BCA assay kit (KeyGen Biotech). Equal amounts of protein were subjected to 10% SDS-PAGE for electrophoresis and transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA) with the use of a Transblot apparatus (Bio-Rad, Hercules, CA, USA). The membranes were blocked at room temperature for 1 h with 5% bovine serum albumin and then incubated with anti-TCF21 antibody (ab182134, 1:1000 dilution; Abcam), anti-SF-1 antibody (ab168380, 1:1000 dilution; Abcam), and anti-GAPDH antibody (1:1000 dilution; ZSGB-BIO, Beijing, China) at 4 °C overnight. The next day, the membranes were incubated with the appropriate secondary antibodies at a 1:5000 dilution for 1 h at 37 °C. Protein bands were visualized by enhanced chemiluminescence (ECL) solution (Syngene). The intensities of the western blot bands were analyzed using ImageJ software and normalized with respect to GAPDH. Independent experiments were performed using cultured cells from at least three different subjects and repeated three times.