Igepal np 40

Chemicals were obtained from the following sources: $\text{β}$ -estradiol (E8875), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (C2920), cOmplete Protease Inhibitor Cocktail (11697498001), dimethyl sulfoxide (D2650), cycloheximide (C1988), doxycycline hyclate (C9891), polyvinylpyrrolidone (PVP40), pepstatin (10253286001), phenylmethylsulfonyl fluoride (P7626), calcofluor Fluorescent Brightener 28 (F3543) from Millipore Sigma, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (D130), ProLong Glass Antifade Mountant with NucBlue Stain (P36981) from ThermoFisher, (S)-MG-132 (10012628) from Cayman Chemical, N-ethylmaleimide (NEM) (S3876), IGEPAL NP-40 (CA-630) from Sigma–Aldrich, Zymolyase 100T (Z1004) from US Biological Life Sciences, Triton X-100 (1610407) from Bio-Rad, paraformaldehyde (100503–914) from VWR, and dithiothreitol (DTT10) from GOLDBIO. Antibodies and other reagents are described in the appropriate section below.

Frozen tissue (100 mg) was manually homogenised in ~ 1:10 (w/v) Igepal/NP-40 (Sigma, Dorset, UK)-based lysis buffer (in mM: 20 HEPES, 150 NaCl, 1% Igepal/NP-40, 0.1 EDTA, pH = 7.6) including protease and phosphatase inhibitors (cOmplete mini and PhosStop, Roche Life Science, Burgess Hill, UK). Homogenates were centrifuged (13,000g, 4 °C, 20 min) and supernatants containing soluble material were stored at − 80 °C.