Protein g plus agarose bead

Cells were lysed in lysis buffer (Cell Signaling, 9803) supplemented with a protease cocktail (Roche, 04693132001). Proteins (15–35 μg) were resolved using 10–12% SDS–PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, IPVH00010). The membranes were blocked with 5% skim milk (Bioshop, SK1400) and then probed with primary and secondary antibodies. The proteins were visualized using a chemiluminescent substrate (Millipore, WBKLS0100). For IP, cells were lysed in lysis buffer (0.05 M Tris-HCl [pH 7.4], 250 mM NaCl, 0.25% Triton X-100, 10% glycerol) with protease cocktail. Total proteins (600 μg) were precleared using 50% protein G Plus-agarose beads (Santa Cruz, sc-2002) and centrifuged at 12,000 × g for 10 min at 4 °C. The supernatants were incubated with the indicated primary antibody, rabbit IgG (Sigma-Aldrich, 12-370), or mouse IgG (Sigma-Aldrich, 12-371) overnight at 4 °C. The immunocomplexes were captured using protein G Plus-agarose beads and washed with ice-cold PBS several times. The washed beads were resuspended in 2X Laemmli loading buffer and boiled, and then the proteins were eluted and processed for immunoblotting. The band intensities were quantified using ImageJ software.

The cell lines samples were prepared for IP as indicated in the “Immunoblotting” section. For purification of CERS6 (Fig. 5a), 2000 µg of protein lysates as prepared above were pulled down at 4 °C overnight with Ni-NTA His-Bind Resin (EMD-Millipore, Billerica, MA), washed 4 times with modified RIPA and then eluted with 350 mM imidazole. Further, the eluate was incubated with EZview Red Streptavidin Affinity Gel (Sigma) for 2 h, washed 4 times with modified RIPA, and digested with AcTEV Protease (Invitrogen, Carlsbad, CA) for 3 h. Immunoprecipitation of Fas was performed using 1000 µg of protein lysates incubated with 2 µg of anti-Fas antibody Rb (Proteintech, Rosemont, IL) overnight at 4 °C. Fas and associated proteins were pulled down using 50 µl of Protein G Plus Agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) and sample prepared using 4× NuPAGE lithium dodecyl sulfate loading buffer and 100 mM DTT, followed by heating at 70 °C for 10 min. For immunoprecipitation of CERS6 (Fig. 6b), 1000 µg of protein lysates were pulled down at 4 °C overnight with 50 µl EZview Red anti-FLAG-M2 affinity gels (Sigma-Aldrich), washed 4 times with modified RIPA and then eluted with an excess of 3× FLAG peptide (100 μg/ml). Immuno-complexes were resolved by 4–12% SDS-PAGE and immunoblotted with the indicated antibodies.

HeLa cells were transfected with the different myc-tagged human mastermind constructs (MAML1-3) and HA-Ub using lipofectamine 2000 in 6-well dishes following the standard protocol. After 24 hours, the transfected cells were lysed in 1ml RIPA buffer (50 mM Tris-Cl, pH 7.9, 150 mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, 1X HALT protease cocktail, and 1% PMSF). Lysates were briefly sonicated and centrifuged to remove cell debris. The supernatants were transferred to new tubes and pre-cleared with Protein G PLUS-Agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for one hour at 4°C. Lysates were then centrifuged, supernatants transferred to a new tube, and 5 μg of anti-myc (9E10) antibody was added and incubated over night at 4°C on a nutator. The next day, protein G beads were added to each tube for one hour for immunoprecipitation. Beads were centrifuged and washed three times with RIPA buffer. After removal of the last wash, the beads were prepared for SDS-PAGE by adding 2X Laemmli buffer and boiling for 5 minutes prior to running on 4–12% Bis-Tris Gels (Invitrogen) or 10% SDS gels[27 (link)].

Cells were lysed in culture dishes by adding 0.5 mL of ice-cold RIPA (radioimmunoprecipitation assay) lysis buffer for 45 min. The supernatants were collected by centrifugation at 12,000 g for 15 min at 4°C and then precleared with the protein G PLUS-Agarose beads (Santa Cruz) to remove the non-specific protein G-bounded proteins. The cleared lysate was then incubated with FD10 (5 μg/mL) at 4°C overnight, followed by incubation with the protein G PLUS-Agarose beads for 4 h. Immunoprecipitates were washed 3 times with lysis buffer and then analyzed by immunoblotting.