Western Blot Analysis of CACNA1C and SRF Expression

Protein extract preparation from tissue samples and western blotting were performed as described previously [21 (link)]. Primary antibodies against the following antigens were used: CACNA1C (guinea pig, 1:1000, Alomone Labs, Jerusalem, Israel), SRF (rabbit, 1:500, Santa Cruz Biotechnology, Dallas, TX), or GAPDH (rabbit, 1:1500, Cell Signaling Technology, Danvers, MA). All antibodies were incubated in 5% Blotto, non-fat dry milk (Santa Cruz Biotechnology, Dallas, TX). HRP-conjugated goat anti-guinea pig (AP108P), goat-anti-rabbit (AP307P), or goat anti-mouse (AP308P) secondary antibodies (1:50,000; EMD Millipore, Billerica, MA) were used with an enhanced chemiluminescence method (Amersham ECL Advantage, GE Healthcare Life Sciences, Pittsburgh, PA). The protein bands were captured using a CCD-camera system (EC3 410 Imaging System; UVP, Upland, CA) and analyzed with VisionWorksLS software (Version 6.8; UVP, Upland, CA). The expression level of CACNA1C and SRF was normalized to the ratio of CACNA1C and SRF area density divided by GAPDH area density. For quantitative measurements, the values of relative expression for CACNA1C and SRF in KO5D, KO10D, and KO15D small intestine were compared to the expression in WT10D small intestine (set to 1.0).

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Lee M.Y., Park C., Ha S.E., Park P.J., Berent R.M., Jorgensen B.G., Corrigan R.D., Grainger N., Blair P.J., Slivano O.J., Miano J.M., Ward S.M., Smith T.K., Sanders K.M, & Ro S. (2017). Serum response factor regulates smooth muscle contractility via myotonic dystrophy protein kinases and L-type calcium channels. PLoS ONE, 12(2), e0171262.

Publication 2017

CACNA1C GAPDH Blotto, non-fat dry milk VisionWorksLS software (Version 6.8

Antibodies Antigens Chemiluminescence Gapdh Goat Guinea pig Milk Mouse Protein Rabbit Small intestine Tissue

Corresponding Organization : University of Rochester

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Variable analysis

independent variables

Genotype (WT10D, KO5D, KO10D, KO15D)

dependent variables

Expression level of CACNA1C
Expression level of SRF

control variables

Protein extract preparation from tissue samples
Western blotting protocol
Primary antibodies against CACNA1C, SRF, and GAPDH
Secondary antibodies (HRP-conjugated goat anti-guinea pig, goat-anti-rabbit, or goat anti-mouse)
Enhanced chemiluminescence method
Protein band capture and analysis using CCD-camera system and VisionWorksLS software

controls

Positive control: WT10D small intestine (expression levels of CACNA1C and SRF set to 1.0)
Negative control: Not explicitly mentioned

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