Journal: bioRxiv

Article Title: Isoforms of the transcriptional cofactor SIN3 differentially regulate genes necessary for energy metabolism and cell survival

doi: 10.1101/2021.12.31.474661

Figure Lengend Snippet: A , western blot analysis of whole cell extracts prepared from S2 and 187HA cells. Knockdown (KD) of Sin3A in S2 cells results in a decrease in the level of the dominant isoform, SIN3 220. Expression of HA-tagged SIN3 187 was induced via a metallothionine promoter by the addition of CuSO 4 . Blots were probed with SIN3 pan antibody that identifies all SIN3 isoforms or HA antibody to identify SIN3 187HA. α-Tubulin is used as the loading control. B-D , qRT-PCR analysis of Mgst1 (B), TFAM (C) , and Cyt-c-p (D) . Values indicated represent fold change for the specific SIN3 isoform perturbation, SIN3 220 knockdown or SIN3 187 overexpression, over GFP knockdown. Data are from a minimum of three biological replicates. Statistical comparisons between all conditions were done and significant changes are shown in the figure. Error bars represent standard error of the mean. p-value * p<0.05, ** p<0.01, *** p< 0.001 E , Western blot for cyt-c-p of whole cell extracts from indicated cells. α-Tubulin is used as the loading control.

Article Snippet: Primary antibodies used were SIN3 (1:1000) , α-tubulin (1:1000, incubated overnight in 5% BSA in 1 X PBST at 4°C, Cell Signaling), HA-HRP (1:6000, Sigma), cyt-c-p (1:100, incubated overnight at 4°C, Thermo Fisher Scientific).

Techniques: Western Blot, Knockdown, Expressing, Control, Quantitative RT-PCR, Over Expression