Ei9051

Protein extracts were prepared using RIPA buffer (50 mM/l Tris–HCl pH 7.4, 150 mM/L NaCl, 0.1% sodium dodecyl sulfate (SDS; w/v), 1% sodium deoxycholate (w/v), and 1% Triton X-100 (v/v)) and analyzed by western blotting as previously described (43–45 (link)). Briefly, protein samples were denatured at 100°C for 5 min in the presence of protein loading buffer (62.5 mM Tris–HCl pH 6.8, 10% glycerol (v/v), 0.01% bromophenol blue (w/v), 2.15% SDS (w/v), 1.55% dithiothreitol (w/v), and 5% 2-hydroxy-1-ethanethiol (v/v)), separated on 12% SDS-PAGE gels, and transferred to polyvinylidene fluoride (PVDF) membranes (BioRad, 1620177). Membranes were then processed following the standard ECL protocol (Thermo Fisher Scientific, EI9051). Blots were viewed using Bio-Rad ChemiDoc Imaging System and protein levels were quantified from at least three western blots using ImageJ. Antibodies used were anti-FLAG (Beyotime, AF519; 1:1000 dilution), anti-HDAC1 (Abcam, ab1767; 1:1000 dilution), anti-α-Tubulin (Beyotime, AF0001; 1:2000 dilution), and anti-Histone H3 (Abcam, ab1791; 1:1000 dilution).

Whole-cell protein extracts were prepared using RIPA buffer, separated on NuPAGE 4%–12% Bis-Tris gels (Thermo Fisher Scientific, NP0323), and transferred to a PVDF membrane (Bio-Rad, 1620177). Antibodies used were α-Flag M2 antibody (1:1,000 dilution; Sigma, F1804), α-Tubulin antibody (1:10,000 dilution; Sigma, T6074), and α-Discs Large (1:1,000 dilution; Developmental Studies Hybridoma Bank, 4F3). Membranes were processed using a standard ECL protocol (Thermo Fisher Scientific, EI9051).

Protein extracts from nuclear fractions, cytoplasmic fractions, whole cells or IP samples were subjected to western blotting analyses. The same amount of protein from each sample was separated on NuPAGE 4–12% Bis–Tris gel (Thermo Fisher Scientific, NP0329BOX). The gel was then transferred to polyvinylidene fluoride membranes (Bio-Rad, 1620177). Membranes were processed following the ECL western blotting protocol (Thermo Fisher Scientific, EI9051), as described previously (37 (link),47 (link)). These antibodies were used: anti-HDAC1 (Abcam, ab1767, 1:1000 dilution), anti-α-Tubulin (Sigma, T6074, 1:10 000 dilution), anti-Lam (Beyotime, AF5222, 1:300 dilution), anti-FLAG (Beyotime, AF519, 1:1000 dilution), anti-HA (Beyotime, AF0039, 1:1000 dilution), anti-gawky (anti1, 1:250 dilution; anti2*, 1:250 dilution), and anti-MTF-1 (1: 500 dilution). Blots were viewed with a Bio-Rad ChemiDoc Imaging System and quantified using ImageJ software. * Anti-gawky2 (anti2) was purified from rabbit antisera, raised against amino acids 928–941 of gawky conjugated to KLH. Considering that the specificity of anti1 is better than anti2, anti2 was only used to confirm the knockdown efficiency of gawky dsRNAs.