Protein Extraction and Western Blot Analysis

Protein was extracted using RIPA lysis buffer (Strong, YEASEN) and electrophoresis was performed using a PAGE Gel Quick Preparation Kit (10%, YEASEN) following the manufacturer's instructions. WB was carried out with the following antibodies: (primary) RARA (Invitrogen, #MA1-810A, 1:1000), RXRA (Invitrogen, #433900, 1:1000), HOXA1 (Invitrogen, #PA5-68809, 1:1000), FLAG (Beytime, #AF-0036, 1:1000) and GAPDH (Santa Cruz, #SC-365062, 1:2000), and then HRP-linked secondary antibodies (Sungene Biotech). HRP activity was detected using Luminol HRP Substrate (Millipore). Digital images were taken using an automatic chemiluminescence imaging analysis system (Tanon) (6 (link),26 (link),32 (link),33 (link)).

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Su G., Wang W., Zhao X., Chen J., Zheng J., Liu M., Bi J., Guo D., Chen B., Zhao Z., Shi J., Zhang L, & Lu W. (2021). Enhancer architecture-dependent multilayered transcriptional regulation orchestrates RA signaling-induced early lineage differentiation of ESCs. Nucleic Acids Research, 49(20), 11575-11595.

Publication 2021

RIPA lysis buffer (Strong PAGE Gel Quick Preparation Kit GAPDH HRP-linked secondary antibodies Luminol HRP Substrate

Antibodies Buffer Chemiluminescence Electrophoresis Gapdh Gel preparation Luminol Protein Ripa

Corresponding Organization : Nankai University

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Variable analysis

independent variables

RIPA lysis buffer (Strong, YEASEN) used for protein extraction
PAGE Gel Quick Preparation Kit (10%, YEASEN) used for electrophoresis

dependent variables

Protein expression levels of RARA, RXRA, HOXA1, FLAG, and GAPDH as detected by Western blotting

control variables

Manufacturer's instructions were followed for protein extraction and electrophoresis
GAPDH was used as a loading control

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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