P8430

Cytosol/membrane fractionation assay was performed according to a previously described method (Baghirova et al., 2015 (link)). Briefly, HEK 293T cells were harvested by trypsin treatment and washed with ice-cold PBS. Fractionation buffer FA (150 mM NaCl, 50 mM HEPES pH 7.4, 100 μg/ml digitonin (D141, Sigma), and 1 M hexylene glycol) supplemented with 1x protease inhibitor cocktail (P8340, Sigma, 100x stock solution) was added to the harvested cells to release cytosolic proteins. After incubation for 15 min on an end-to-end rotator at 4 ˚C, samples were centrifuged at 2,000 g for 10 min. The resulting supernatant was transferred and marked as cytosolic proteins. Fractionation buffer FB (150 mM NaCl, 50 mM HEPES pH 7.4, 1% (v/v) Nonidet-P40, and 1 M hexylene glycol) supplemented with 1x protease inhibitor cocktail (P8430, Sigma, 100x stock solution) was added to the resulting cell pellets to release membrane proteins. After incubation for 30 min on ice, samples were centrifuged at 7,000 g for 10 min. The resulting supernatant was transferred and marked as membrane proteins. Both cytosolic and membrane fractions were further immunoprecipitated using a FLAG-M2-affinity gel (A2220, Sigma, RRID:AB_10063035) and analyzed by immunoblotting. These experiments were repeated twice.

Fibroblasts cultured on elastic surfaces were trypsinized and pelleted prior to lysis. Cell pellets were lysed with RIPA lysis buffer (1% NP-40; 0.5% NaDOC; 0.1% SDS; 50 mM Tris–HCl, pH 7.4; 150 mM NaCl; 10% glycerol; 1 mM EDTA) supplemented with protease inhibitors (Sigma, P8430) and phosphatase inhibitors (10 mM NaF; 1 mM Na₃VO₄; 1 mM EGTA). Lysates were incubated on ice for 30 min with periodic vortexing; this was followed by brief sonication and centrifugation at 16 000 × g for 15 min at 4 °C. Cleared lysates were then transferred to fresh microcentrifuge tubes for total protein determination by BCA assay (Thermo Scientific, # 23,225). Cytoplasmic and nuclear cell fractionation was achieved using the Pierce NE-PER™ Extraction reagents (Thermo Scientific, #78,833). All samples were kept at –20 °C for short-term use, or –80 °C for long-term storage (Table 1).Table 1

siRNA oligo pools

Target	Species	Accession	Pool (Dharmacon™ Cat#)
Lats1	R. norvegicus	NM_001134543.2	M-080189–01-0005
Lats2	R. norvegicus	NM_001107267.1	M-087043–01-0005
Limd1	R. norvegicus	NM_001112737.2	L-081750–02-0005
Ski	R. norvegicus	XM_017593893	L-099478–02-0005
Wwtr1 (Taz)	R. norvegicus	NM_001024869.1	M-088521–01-0005
Non-targeting pool	H. sapiens, M. musculus, R. norvegicus	n/a	D-001810–10-05