TBX18-ADSCs and GFP-ADSCs co-cultured with NRVMs and NRVMs cultured alone were plated on 6-well culture dishes. The cells were harvested using RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Equal amounts of protein were loaded onto a gel for sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and the separated proteins were transferred to a nitrocellulose membrane, and then incubated with the primary antibodies against HCN4 (ab32675; Abcam, Cambridge, MA, USA) overnight at 4°C. The primary antibodies were detected by incubating the membrane with horseradish peroxidase-conjugated secondary antibodies (KPL, 14-16-06, 074-1506) raised in the appropriate species, and then performing enhanced chemiluminescence detection (ECL; Beyotime Institute of Biotechnology). The level of GAPDH was used to normalize the signal intensities.
Ab32675
Manufactured by Abcam
Sourced in United States
Ab32675 is a laboratory equipment product. It is a scientific instrument used for [core function]. The product specifications and technical details are available on the Abcam website.
Automatically generated - may contain errors
3 protocols using ab32675
1
Quantification of Cardiac Ion Channel Expression
2
Protein Expression Analysis of Cardiac Progenitors
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The differentiated hiPSCs on D20 were plated on 12-well culture dishes. The cells were harvested using RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Equal amounts of proteins were loaded onto a gel for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and the separated proteins were transferred onto a nitrocellulose membrane, and incubated with primary antibodies against HCN4 (ab32675; Abcam, Cambridge, MA, USA), TBX18(ab115262; Abcam, Cambridge, MA, USA), ISL-1(abs132916; Abcam, Cambridge, MA, USA) TBX3(ab154828; Abcam, Cambridge, MA, USA), TBX5(Gene-Tex; Cat No. GTX113849), α-tubulin (GB11200; Service-bio, Wuhan, China), GAPDH antibody (Abcam; Cat. no. ab37168) and Shox2(ab55740; Abcam, Cambridge, MA, USA) overnight at 4 °C. The primary antibodies were detected by incubating the membrane with secondary antibodies for 1 h, and then enhanced chemiluminescence detection (ECL; Beyotime Institute of Biotechnology) was performed. The level of α-tubulin or GAPDH was considered to normalize the signal intensities. The Western blot assay was repeated at least three times to verify the results.
3
Immunofluorescence Staining of HCN4 in Cultured Cells
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A total of 1 week post-transduction, immunofluorescence staining was performed on cultured cells. Briefly, approximately (5 (link)-8 (link))x105 cells grown on glass cover-slips were fixed with 4% paraformaldehyde for 20 min at room temperature. Following permeabilization with 0.1% Triton X-100, cells were incubated overnight with rat anti-HCN4 monoclonal primary antibody (ab32675, 1:50; Abcam) at 4°C and were then incubated with Cy3-labeled goat anti-rat secondary antibody (AS-1111, 1:50; Aspen Biotechnology, Hubei, China) for 50 min at room temperature. Nuclei were also stained with 4′,6-diamidino-2-phenylindole for 5 min. Fluorescent images were acquired using an inverted fluorescent microscopy.
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