Mouse anti lgbit

Relative expression of mG probes was assessed by Western blot and in-gel fluorescence. HeLa cells transfected with LgBiT-mG or mRuby2-mG probes were lysed in RIPA buffer and sonicated, and total protein amounts were quantified. Western blot assays followed the procedure described under the “ERK1/2 activation assay” section. Membranes were incubated overnight at 4°C with mouse anti-LgBiT (1:500; Promega, catalog no. N710A) or goat anti-mRuby (1:2000; St John’s Laboratory, catalog no. STJ140251) primary antibodies in TBS-Tween (0.1% v/v) with 5% BSA. After washing, HRP-coupled anti-mouse and anti-goat (1:10,000; Jackson ImmunoResearch, catalog no. 705-035-003) secondary antibodies were added for 1 hour at room temperature, and the membranes were imaged as described above. Tubulin, probed with a recombinant mouse IG2A anti-alpha tubulin antibody (1:5000; Geneva Antibody Facility, ABCD_AA345), served as loading control. For in-gel fluorescence, lysates of HeLa cells expressing mRuby2-mG probes were mixed with NuPAGE LDS sample buffer and loaded directly on a 4 to 12% bis-tris plus gel for SDS-PAGE separation. Gels were then imaged with the Odyssey M (LI-COR) reader, and mRuby2 was detected at 520 nm excitation. Proteins were transferred to an isopropanol-activated PVDF membrane and stained with Ponceau S as loading control.