Western Blot Analysis of Apoptosis Markers

Total proteins were isolated using radioimmunoprecipitation lysis buffer (Beyotime) and loaded onto SDS-PAGE gel for separation and then transferred to PVDF membranes (BioRad, 1,620,177). After that, the membranes were blocked with skim milk for 2 h, incubated with primary antibodies against MYRF (1:1000; Abcam, ab227721), cleaved-Caspase-3 (1:1000; Abcam, ab32499), Bax (1:1000; Abcam, ab182733), BCL2 (1:1000; Abcam, ab182858), and GAPDH (1:1000; Abcam, ab9485) at 4°C overnight. Then an enhanced chemiluminescence kit (Santa Cruz, sc-2048) was employed to detect the protein signals following another 2 h incubation with HPR-conjugated secondary antibody. Protein levels were quantified using Image-Pro® Plus software (Media Cybernetics) [18 (link)].

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Yang X., Wang L., Zhou F., Ye S, & Sun Q. (2022). Yin Yang 1-induced activation of LINC01133 facilitates the progression of pancreatic cancer by sponging miR-199b-5p to upregulate myelin regulatory factor expression. Bioengineered, 13(5), 13352-13365.

Publication 2022

radioimmunoprecipitation lysis buffer PVDF membranes ab32499 ab182733 ab182858 ab9485 enhanced chemiluminescence kit sc-2048 Image-Pro® Plus software

Antibodies Antibody Bcl2 Buffer Caspase 3 Chemiluminescence Gapdh Membranes Milk Protein Pvdf Radioimmunoprecipitation Sds page

Corresponding Organization : Xuzhou Medical College

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Variable analysis

independent variables

Radioimmunoprecipitation lysis buffer (Beyotime)

dependent variables

Protein levels of MYRF, cleaved-Caspase-3, Bax, BCL2, and GAPDH

control variables

GAPDH

controls

Positive control: Not specified
Negative control: Not specified

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