Briefly, total protein of LUSC tissue samples and cell lines was extracted using protein extraction reagent (Thermo Scientific) with a cocktail of proteinase inhibitors (Roche Applied Science, Switzerland) and a cocktail of phosphatase inhibitors (Roche Applied Science) according to its protocol. Equal amount of total protein (20 μg) was separated by 10% SDS–PAGE and transferred onto a PVDF membrane. After blocking for nonspecific binding, the membranes were incubated with antibody FOXM1 (1:3000 dilution; proteintech; 13147-1-AP), CENPA (1:1000 dilution; Abcam; ab45694), CENPB (1:1000 dilution; Abcam; ab25734), CCNB1 (1:1000 dilution, Cell Signaling Technology; #4134), or β-actin (1:10000 dilution; Sigma; A2228) overnight at 4 °C and followed by an incubation period of 1 h at room temperature with secondary antibody (1:4000, Bioword; 20330016-1). Bands were detected by a Bio-rad ChemiDoc XRS system. Full scans of the western blots shown in Figs. 4 e, f, 5 g and 6a, b and Supplementary Figs. 5 c and 6c are provided in Source Data file.
Ab45694
Manufactured by Abcam
Sourced in United Kingdom
Ab45694 is a lab equipment product. It is designed to perform a core function in a laboratory setting. No further details are available for this product at this time.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using ab45694
1
Western Blot Analysis of LUSC Proteins
2
Western Blot Analysis of Cell Proteins
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The cell or tumor tissue precipitate was lysed in lysis buffer. The homogenates were put on ice for 10 m and centrifuged (3,000 g, 4°C, 10 m). The supernatant was transferred to a precooled Eppendorf tube. Following protein concentration determination by BCA kits, the proteins were transferred to a PVDF membrane and blocked with 5% BSA at room temperature for 1 h and incubated with antibodies against CENPA (ab45694, 1:1,000; Abcam, Cambridge, UK), STMN1 (ab52630, 1:10,000; Abcam), and ACTIN (ab8226, 1:1,000; Abcam) overnight at 4°C. The next day, the proteins were incubated with the secondary antibody at room temperature for 1 h, followed by development on the gel imaging system. The protein bands were subjected to grayscale scanning with AlphaView SA software (Version: 3.4.0).
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