Western Blot and Immunofluorescence Staining Protocol

Rabbit anti-SOX7 (BA3297-2, diluted at 1:1000 for WB (western blotting assay)) was obtained from Boster (Wuhan, China), rabbit anti-VE-cadherin (#2500, diluted at 1:1000 for WB and 1:200 for IF (immunofluorescence staining) and rabbit anti-GAPDH (#5174S, diluted at 1: 1000 for WB) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). miR-21-5p mimics (miR10000076), miR-21-5p inhibitor (miR20000076) were purchased from RiboBio Co., Ltd., China.
miR-21-5p mimics are chemically synthesized miRNA which is easy to control transient transfection into the cell, while miR-21-5p inhibitor is single-stranded synthetic inhibitor having complementary sequences to target miRNA.
PM2.5 were sampled from Beijing and analyzed as our previous research [7 (link)].

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Wang S., Lin Y., Zhong Y., Zhao M., Yao W., Ren X., Wang Q., Guo X., Zhang Q.Q, & Dai J. (2020). The long noncoding RNA HCG18 participates in PM2.5-mediated vascular endothelial barrier dysfunction. Aging (Albany NY), 12(23), 23960-23973.

Publication 2020

rabbit anti-VE-cadherin rabbit anti-GAPDH (#5174S, miR-21-5p mimics miR-21-5p inhibitor

Cell Gapdh Immunofluorescence Mirna Rabbit Transfection Transient Ve cadherin Western blotting assay

Corresponding Organization :

Other organizations : Third Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Beijing Haidian Hospital, Hebei Agricultural University, Chinese Center For Disease Control and Prevention, Guangdong Pharmaceutical University

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Variable analysis

independent variables

MiR-21-5p mimics
MiR-21-5p inhibitor

dependent variables

SOX7 protein expression (measured by Western blotting)
VE-cadherin protein expression (measured by Western blotting and immunofluorescence staining)

control variables

GAPDH protein expression (measured by Western blotting)

controls

Positive control: Not specified
Negative control: Not specified

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