Extended Data Fig. 6: Detection of CREB, pCREB, and CRTC1.

a. 15 minutes after membrane depolarization is the timepoint at which the greatest number of hGNs immunostain positive using the phosphorylated ser133 CREB antibody used for ChIPseq. Median and interquartile range of counts from six images for each condition from a single immunostaining event of one differentiation lot of 1G neurons. b. Western blot for CRTC1 and GAPDH from hGN cell lysates after 6 or 9 days of non-targeting (NT) or CRTC1-targeting siRNA blotted with the antibody that was used for CRTC1 ChIPseq. Blot displays the two independent experiments performed for this quantification. 9 day treatments were used for siRNA treatments in this study, which resulted in an average 64.1% protein knockdown. c. Western blot of hGN cell lysates using the CREB antibody used for CREB ChIPseq. Image is representative of two independent experiments. d. Immunostaining for CRTC1 in hGN cultures before (unstimulated) and 15 minutes after membrane depolarization. Membrane depolarization causes CRTC1 to translocate to the nucleus. Scale bar = 100 µm. Images are representative of 3 independent experiments.