Figure 3

Solubilization and extraction of nuclei and insoluble proteins. (A) HEK293 cells were plated at a density of 4 × 10⁵ cells per well of a 12 well plate and processed according to the protocol in Figure 2. An aliquot of each extract was then analyzed by 4-12% SDS PAGE followed by staining with Coomassie blue or by Western blotting and probing with antibodies to the nuclear membrane protein Lamin A (SIGMA #L1293) or the ER membrane protein TRAM both highlighted by arrowheads. (B) Extracts generated in (A) were analyzed by 8 or 12% SDS PAGE followed by Western blotting and probing with antibodies to various cytosolic; Akt, Cell Signaling Technology (CST #9272), GSK-3β, (CST #9315), Caspase-3 (CST #9662) and γ-Tubulin (Santa Cruz Biotechnology, sc-7396), Organellar; Calnexin (SIGMA #C4731), Calreticulin (Stressgen #SPA-061), VDAC/Porin (SIGMA #V2139) and nuclear; p53 (CST #9282), RAS (Upstate Biotechnology #05-516) and Histone H3 (ABCAM #ab1791) proteins as indicated or with anti V5 antibody (Invitrogen #R96025) in the case of ERGIC-53. (C) Equal volumes of extracts were analyzed by 4-12% SDS-PAGE followed by staining with Coomassie blue and then silver staining. The bracket alongside the Coomassie stained gel shows the position of Histones in the RIPA nuclear extract and the asterisk next to the silver stained gel denotes a protein band unique to the E-RIPA extract. (D) The subcellular fractionation protocol was carried out in full and in duplicate. In one experiment the RIPA and E-RIPA extracts were prepared as a single extract as described in step 5 of the protocol (Combined). In both cases following the E-RIPA extract any remaining pellet was extracted by boiling in LSB + DTT.