Anti gapdh ab9484

Cell pellets were lysed in Laemmli buffer ×2 (Tris–HCl 0.5 M, pH 6.8; 0.5 M DTE; 0.5% SDS) and protein concentrations determined using the Bradford reagent (Bio-Rad). Protein extracts were separated on SDS–PAGE (8% or 15% or 4–15% gradient [vol/vol]) gels. Gels were transferred onto nitrocellulose membranes (GE HealthCare and Bio-Rad) for immunoblotting with the following antibodies: anti-NLRP3 (Cryo-2, 1:1,000) and anti-caspase-1 (Bally-1, 1:1,000) from AdipoGen, anti-ASC (1:2,000) from Enzo Life Science, and anti-γH2AX (JBW301, 1:1,000), anti-P-Ser15-p53 (1:1,000), and anti-ATM Ser1981 (10H11.E12, 1:1,000) from Millipore. Anti-P-KAP1 Ser824 (1:1,000), anti-KAP1 (1:1,000), and anti-NEK7 (A302-684A, 1:1,000) from Bethyl Laboratories, anti-p53 (clone DO7 1:2,000) and anti-NOXA (114C307, 1:1,000) from Santa Cruz; anti-ATM (#ab32420, 1:1,000), anti-fibrillarin (ab4566, 1:1,000), and anti-GAPDH (ab9484, 1:1,000) from Abcam; anti-FLAG (F7425 1:5,000) and anti-α-tubulin (clone B-5-1-2 1:1,000) from Sigma-Aldrich; anti-XPO2 (GTX102005 1:1,000) and anti-IPO5 (GTX114515 1:1,000) from Genetex, and anti-actin (C4, 1:100,000) from MP Biomedical. The Fiji and Image Laboratory (Bio-Rad) programs were used for densitometric analysis of immunoblots, and the quantified results were normalized as indicated in the figure legends.

Cells were planted to 6-well plates prior to collection for western blotting. The cellular protein samples were obtained from RIPA buffer (Invitrogen), separated by 12% SDS-PAGE gels, and then transferred onto PVDF membranes. 5% skimmed milk was applied to block membranes. Primary antibodies including anti-NOTCH-1 (ab52627), anti-E-cadherin (ab40772), anti-N-cadherin (ab98952), anti-Vimentin (ab92547), anti-ZEB1 (ab81972), anti-ZEB2 (ab138222), anti-EGFR (ab52894), anti-p-EGFR (ab40815), and anti-GAPDH (ab9484), as well as HRP-labeled IgG secondary antibodies, were purchased from Abcam (Cambridge, UK). Primary antibody anti-MDR-1 was obtained from Cell Signaling Technology (#13978, Massachusetts, USA). After washing in phosphate-buffered saline (PBS), membranes were immersed in ECL Prime Western Blotting Detection reagent (GE Healthcare, Chicago, IL, USA) and observed in the dark.