Hrp conjugated affinipure goat anti rabbit igg h l

Placental tissues were homogenized with a tissue grinder and placed in protein lysis buffer (990 µL of RIPA and 10 µL of PMSF). PMSCs were similarly collected, resuspended in protein lysis buffer, and incubated on ice for 30 min. The lysates were centrifuged at 4°C and 12 000 r/min for 30 min, and the supernatant was collected as the protein sample. Nuclear and cytoplasmic proteins were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagent (78835, Thermo, USA). Protein concentrations were determined using a Pierce BCA Protein Assay Kit (23225, Thermo, USA). For each sample, 30 µg of protein was mixed with 5× protein loading buffer containing DTT at a 4:1 volume ratio, boiled for 10 min, and then stored at −80°C. Proteins were separated using 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) based on molecular weight. After electrophoresis, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes, which were blocked at room temperature in 5% skim milk solution (2.5 g of skim milk powder and 50 mL of TBST) for 1 h. The membranes were then incubated overnight at 4°C with diluted primary antibodies (see Supplementary Table S5 for details). After discarding the primary antibodies, the membranes were washed four times (5 min each) with 1× TBST wash buffer on a rocking platform, then incubated with diluted secondary antibodies, including HRP-conjugated AffiniPure goat anti-mouse IgG (H+L) (SA00001-1; 1:5 000, Proteintech, China) and HRP-conjugated AffiniPure goat anti-rabbit IgG (H+L) (SA00001-2; 1:5 000, Proteintech, China), at room temperature on a rocking platform for 1 h. After the secondary antibodies were discarded, the membranes were washed three times (10 min each) in 1× TBST wash buffer on a rocking platform. The chemiluminescent substrate was prepared by mixing luminol reagent and peroxide solution at a 1:1 ratio (32209, Thermo, USA) and applied evenly to the membrane surface. The membranes were then enclosed in self-sealing bags, ensuring the removal of any air bubbles. Exposure was performed using a chemiluminescent gel imaging system. Images were saved in the desired format, and grayscale quantification was performed using ImageJ software.

HEK293T cells were plated in six-well plates. At 70%–80% confluency after plating, cells were transfected corresponding plasmid. After 24 h, cells were washed with phosphate-buffered saline (PBS) once and lysed with 200 μl of 1× SDS Loading buffer (10 mM Tris–HCl, pH 8.0, 50 mM DTT (Dithiothreitol), 1% SDS, 10% glycerol, and 0.008% bromophenol blue) at room temperature for 10 min and then denatured at 95°C for 10 min. The appropriate amount of protein was loaded onto SDS–PAGE gels. After separating proteins by running gel at a constant voltage of 100 V for 1.5 h, proteins were transferred from gel onto a PVDF (Polyvinylidene Fluoride) membrane (Millipore) in an ice-bath for 2 h. Then, the PVDF membrane was blocked in 5% (w/v) BSA (Beijing Dingguo changsheng Biotechnology Co., Ltd, FA016) in TBST (Tris-buffered saline, 0.1% Tween 20) at room temperature for 1 h. The blot of protein was stained as indicate for at least 12 h at 4°C. The blot was washed four times with TBST at room temperature for 5 min each, and then stained with 1:5000 HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (Proteintech, SA00001-2) or HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L) (Proteintech, SA00001-1) in 5% BSA (w/v) in TBST for 1 h at room temperature. The blots were washed four times with TBST at room temperature for 5 min each time and imaged on Molecular Imager ChemiDocTM XRS + Imaging System (Bio-Rad) after incubation with Rhea ECL (US Everbright, Inc).