Western Blot Analysis of Cell Signaling Proteins

Western blot was performed as we recently described [21 (link), 26 ]. Primary antibodies raised against SRSF1 (Santa Cruz, sc-33,652), EIF3B (Santa Cruz, sc-137,214), TIA1 (Santa Cruz, sc-166,247), beta-actin (A5441), and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz, sc-47,724) were used for detection. Nuclear and cytoplasmic protein extraction was extracted following ROCKLAND Nuclear & Cytoplasmic Extract Protocol [34 (link)], and verified by Histone-H3 (CST, 4499S) and GAPDH (Santa Cruz, sc-47,724) detection. Antibodies used for exosome marker detection include: CD63, CD81 (Santa Cruz Bio Technology Inc., CA, USA), Flotillin-1 and Calnexin (Cell Signaling Technology, Inc., MA, USA).

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Xu Y.F., Xu X., Gin A., Nshimiyimana J.D., Mooers B.H., Caputi M., Hannafon B.N, & Ding W.Q. (2020). SRSF1 regulates exosome microRNA enrichment in human cancer cells. Cell Communication and Signaling : CCS, 18, 130.

Publication 2020

EIF3B GAPDH Flotillin-1 Calnexin

Antibodies Beta actin Calnexin Cytoplasmic Exosome Flotillin 1 Glyceraldehyde 3 phosphate dehydrogenase Histone h3 Protein Tia1 Western blot

Corresponding Organization :

Other organizations : University of Oklahoma Health Sciences Center, Florida Atlantic University

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Variable analysis

independent variables

Nuclear and cytoplasmic protein extraction

dependent variables

Detection of SRSF1, EIF3B, TIA1, beta-actin, and GAPDH proteins
Detection of exosome markers CD63, CD81, Flotillin-1, and Calnexin

control variables

Western blot protocol as recently described [21, 26]
Primary antibodies used for detection
Histone-H3 and GAPDH detection for verification of nuclear and cytoplasmic protein extraction

controls

Positive control: Histone-H3 for nuclear protein extraction
Negative control: GAPDH for cytoplasmic protein extraction

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