Nel104001

Cells were lysed in ice-cold radioimmunoprecipitation assay buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1% Na-deoxycholate) with protease inhibitors. Lysate protein concentrations were quantified using the Bradford assay, and 50 μg of lysate was loaded into 10–15% polyacrylamide gels and separated at 125 V for 1.5 h. The resolved proteins were then transferred onto nitrocellulose membranes (or PVDF membranes for ATG12 and LC3B immunoblots) for 2 h at 90 V. Membranes were blocked in 5% nonfat milk in TBS-T (50 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.6) for 1 h at room temperature, washed twice in TBS-T for 5 min, and incubated in primary antibody overnight at 4 °C with constant agitation. Membranes were then washed and incubated for 1 h at room temperature with mouse or rabbit IgG-HRP secondary antibodies (Sigma-Aldrich #A4416 and #A4914) diluted 1:2000 with 5% milk in TBS-T. Finally, membranes were washed and developed using enhanced chemiluminescence (PerkinElmer #NEL104001). When applicable, densitometry was performed using Image Studio Lite (LI-COR Biosciences).

Immunoblot analysis was performed using primary rabbit anti-human EOMES (AB23345) or our custom primary rabbit EOMES PTM antibodies and secondary HRP-conjugated goat-anti-rabbit antibody on nuclear extracts isolated from Jurkat T cells either transfected with EOMES WT plasmids, EOMES mutant 1 plasmid, LSD1 WT plasmid, or LSD1 NLS mutant plasmid. Signals were detected with enhanced chemiluminescence reagents (Western Lightning ECL-Plus; Perkin-Elmer, NEL104001) and film exposure. Band intensity signals were normalized to the total protein transferred to the blot detected using the Quantitative Novex Reversible Protein Stain (Thermo Fisher Scientific, IB7710) and Image J analysis.

Immunoblot analysis was performed using primary mouse antibody to human SC35p (as above, ab11826) and secondary HRP-conjugated goat-anti-mouse antibody on nuclear extracts isolated from either primary human CD4⁺ cells, which were untreated (mock) or treated with Life Technologies siRNA PKC-θ pool (Life Technologies ID s11122, s11123), PKC-θ siRNA (Santa Cruz SC-36252), or nuclear extracts from NS or ST Jurkat T cells either untreated or treated with rottlerin. Nuclear extracts of transfected Jurkat T cells were also probed for nuclear expression of HA-tagged PKC-θ WT or NLS mutant constructs using a primary rabbit antibody to HA (Sigma, H6908). Signals were detected with enhanced chemiluminescence reagents (Western Lightning ECL-Plus, Perkin-Elmer NEL104001) and film exposure. Band intensity signals were normalized to the total protein transferred to the blot detected using the Quantitative Novex Reversible Protein Stain (Thermo Fisher Scientific IB7710) and Image J analysis.