Westerns were performed using standard procedures by running RIPA cell lysates on denaturing polyacrylamide gels, transferring to PVDF membranes, and staining with the following antibodies: SAT1: ab105220 (Abcam); MELK: HPA017214 (Sigma); FOXM1: PA5-27144 (Thermo Fisher); β-actin: A1978 (Sigma). Quantification of the blots was performed with ImageJ, and statistical calculations by student’s t tests were performed with GraphPad Prism 7.05.
Hpa017214
Manufactured by Merck Group
HPA017214 is a laboratory equipment product. It serves as a core function within a research or analytical setting. Detailed technical specifications are not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Ab105220, by Abcam (1 mentions)
Foxm1, by Thermo Fisher Scientific (1 mentions)
A1978, by Merck Group (1 mentions)
Prism 7, by GraphPad (1 mentions)
β actin, by Merck Group (1 mentions)
Ab56382, by Abcam (1 mentions)
Sc 365685, by Santa Cruz Biotechnology (1 mentions)
Ultravision detection system, by Thermo Fisher Scientific (1 mentions)
3 protocols using hpa017214
1
Western Blot Analysis of Protein Targets
2
Immunohistochemical analysis of MELK in LMS
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The LMS TMAs were established by acquiring a 1-mm-diameter core at a representative area in each tumor. The TMA block was built on a semiautomatic tissue arrayer (MiniCore, UK).
Immunohistochemistry was conducted on histological sections of formalin-fixed, paraffin-embedded LMS tissue samples in TMAs. The sections were deparaffinized and rehydrated by xylene and ethanol. Antigen was retrieved with EDTA buffer (pH = 8.0) at 98°C for 15 min. Then, 3% hydrogen peroxide was used to block the endogenous peroxidase, and nonspecific binding was obstructed with goat serum. The primary antibody anti-MELK (1:500 dilution, HPA017214, Sigma Aldrich) was incubated overnight in a humid chamber at 4°C. Expression was visualized by I-View 3,3amberilution, HPA0 staining detection.
Immunohistochemistry was conducted on histological sections of formalin-fixed, paraffin-embedded LMS tissue samples in TMAs. The sections were deparaffinized and rehydrated by xylene and ethanol. Antigen was retrieved with EDTA buffer (pH = 8.0) at 98°C for 15 min. Then, 3% hydrogen peroxide was used to block the endogenous peroxidase, and nonspecific binding was obstructed with goat serum. The primary antibody anti-MELK (1:500 dilution, HPA017214, Sigma Aldrich) was incubated overnight in a humid chamber at 4°C. Expression was visualized by I-View 3,3amberilution, HPA0 staining detection.
3
Immunostaining of Cell Cycle Regulators
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Tissue specimens were immunostained with an Ultra-Vision Detection System (Thermo Scientific) following the manufacturer’s protocol. Primary rabbit polyclonal antibodies against budding uninhibited by benzimidazoles 1 (BUB1; 1:50, sc-365685; Santa Cruz Biotechnology), cyclin-dependent kinase 1 (CDK1; 1:100, HPA003387; Sigma-Aldrich), maternal embryonic leucine zipper kinase (MELK; 1:200, HPA017214; Sigma-Aldrich), and non-SMC condensin I complex subunit G (NCAPG; 1:150, ab56382; Abcam) were used for immunochemistry. The procedures were described previously (Goto et al, 2015 (link); Goto et al, 2016a (link); Goto et al, 2016b (link)).
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