Hrp conjugated goat anti rabbit igg as1107

Cells were lysed with RIPA buffer (Beyotime, Shanghai, China). The protein concentration was quantified using BCA protein assay kit (Beyotime). The following steps were done as mentioned in previous study
[24] (link). Immunoreactive bands were revealed using BeyoECL Plus kit (Beyotime). The images were acquired by using Bio-Rad ChemiDoc™ XRS+System (Bio-Rad, Hercules, USA). Antibodies against the following proteins were used: QKI5 (13169-1-AP, 1:1000 dilution; Proteintech, Rosemont, USA), AKT1 (55230-1-AP, 1:1000 dilution; Sanying, Wuhan, China), β-actin (60008-1-Ig, 1:1000 dilution; Sanying), α-tubulin (ab7291, 1:1000 dilution; Abcam, Cambridge, UK). HRP-conjugated goat anti-rabbit IgG (AS1107, 1:1000 dilution; ASPEN, Wuhan, China) or goat anti-mouse IgG (AS1106, 1:1000 dilution; ASPEN) was used as the secondary antibody.

Cells were harvested, washed in PBS and lysed in extraction buffer containing 1% Nonidet P-40, 0.01% SDS and a protease inhibitor cocktail (Roche Diagnostics). Protein contents were determined using a bicinchoninic acid assay kit (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of each protein extract (20 µg per lane) were resolved using 10% SDS-PAGE. Following transblotting onto Immobilon-P membranes (EMD Millipore) and blocking with 5% nonfat milk in saline buffer for 1 h at room temperature, the membranes were incubated with antibodies to p53 (cat. no. ab179477; Abcam; 1:1,000), TRPM1 (cat. no. ab72154; Abcam; 1:1,500), MMP9 (cat. no. ab76003; Abcam; 1:1,000) and GAPDH (cat. no. ab37168; Abcam; 1:10,000) for 1 h at room temperature. The membranes were then washed and incubated with HRP-conjugated goat anti-rabbit IgG (AS1107; Aspen Biological; 1:10,000) for 1 h at room temperature. Each membrane was then washed again and specific immunoreactive bands were visualized using an enhanced chemiluminescent reaction (ECL kit; Amersham; GE Healthcare). Signal intensities were quantified using ImageJ software version 1.47 (National Institutes of Health) and were normalized to GAPDH. All data were obtained from more than two independent experiments carried out in triplicate.