Anti myc agarose affinity gel

Dbf2 kinase assays were performed as previously described (Visintin and Amon, 2001 (link)) with the following modifications: Immunoprecipitations were performed with an anti-Myc agarose affinity gel (Sigma). Kinase reactions were incubated for 1 hr with gentle mixing and separated by SDS PAGE. Histone H1 phosphorylation was quantified using the phosphorImaging system. Prior to this analysis, the top part of the gel was cut off the kinase assay gel and processed for Dbf2 western blot analysis. Proteins were transferred onto nitrocellulose using semi-dry blotting. The membrane was probed with an anti-c-Myc [9E10] antibody (abcam, 1:500 dilution) and imaged using the ECL Plus system (GE Healthcare).

Cell lysates were prepared in lysis buffer (50 mM Tris-HCl (pH 7.5), 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 10 mM sodium fluoride, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 150 mM NaCl, and 10 mg/ml pepstatin A) containing 1% NP-40. Soluble proteins were subjected to immunoprecipitation with anti-Flag or Anti-Myc Agarose Affinity Gel (Sigma-Aldrich) antibodies. An aliquot of the total lysates (5%, v/v) was included as control. Immunoblotting analysis was performed with anti-Myc, anti-HA, anti-Flag, anti-α-Tubulin (Sigma-Aldrich), anti-HMGB1 (ab92310, Abcam, Cambridge, UK), anti-caspase-1 (sc-514, Santa Cruz Biotechnology), anti-ASC (sc-271054, Santa Cruz), and anti-NLRP3 (AG-20B-0014-C100, Adipogen, San Diego, CA, USA) antibodies, respectively. The antigen-antibody complexes were visualized by chemiluminescence (5200, Tanon, Inc., Shanghai, China). The Band Analysis tools of Gel Image System software version 4.2 (Tanon) were used to select and determine the background-subtracted density of the bands in all the gels and blots. Level of protein was first normalized to respective tubulin control. The control (wild-type) condition was normalized to 1 and all other experimental conditions were compared with this.

Ubiquitination assay was performed as previously described [58 (link)]. GSCs overexpressing shNT or shPML shRNAs were treated with the proteasome inhibitor MG132 (20 μM, Sigma-Aldrich, C2211–5MG) for 6 hours and then lysed in TritonX-100 lysis buffer, immunoprecipitated with anti-Myc agarose affinity gel (Sigma-Aldrich, A7470–1ML) and immunoblotted with an anti-ubiquitin (Biolegend, 646301) or anti-c-Myc antibody (Santa Cruz, sc-40). Briefly, cell lysates (300 μg of total protein) were incubated with 15 μL anti-Myc conjugated agarose gel with constant rotation overnight at 4°C. Immunocomplexes were washed three times with ice-cold 0.3% Triton X-100 in PBS buffer and eluted in loading buffer by boiling for 10 min, and then analyzed by immunoblotting. Proteins were resolved on NuPAGE Novex 4–12% Bis-Tris gels (Invitrogen, NP0322BOX), blotted onto polyvinylidene membranes and probed by antibodies specific to ubiquitin and c-Myc.