Journal: Cell Death & Disease

Article Title: GNA13 regulates BCL2 expression and the sensitivity of GCB-DLBCL cells to BCL2 inhibitors in a palmitoylation-dependent manner

doi: 10.1038/s41419-020-03311-1

Figure Lengend Snippet: A The scheme of isobaric iodoTMT switch labeling-based mass spectrometry assay and results of MS/MS spectrum of palmitoylated peptide of GNA13. IodoTMT 6 -127 labeling on Cys14 and Cys18 (the two C in lowercase) of GNA13 peptide sequence was shown in peaks graph (upper panel). B Total, membrane (Mem) and cytosolic (Cyto) fractions of HeLa cells expressing HA-tagged WT GNA13, C14S, C18S, or C14/18S mutant of GNA13 were immunoblotted with an anti-HA antibody. α-Tubulin was used as a loading control for total cellular proteins, while Na-K-ATPase and GAPDH were used as markers of the membrane and cytosol, respectively. C HeLa cells overexpressing WT GNA13 or C14/18S mutant were incubated with cycloheximide (CHX) and analyzed by western blot at the indicated time points. D Protein levels of WT GNA13 and C14/18S mutant in HeLa cells treated with or without indicated caspase inhibitors for 24 h were detected by immunoblotting with an anti-HA antibody. α-Tubulin was used as a loading control.

Article Snippet: The cycloheximide (CHX), autophagy inhibitors, Hydroxychloroquine sulfate (HCQ), ULK-101, and the pan-caspase inhibitor Z-VAD(OMe)-FMK were purchased from TargetMol (Wellesley Hills, MA, USA).

Techniques: Labeling, Mass Spectrometry, Tandem Mass Spectroscopy, Sequencing, Membrane, Expressing, Mutagenesis, Control, Incubation, Western Blot

Journal: Science Advances

Article Title: Targeting oncoproteins with a positive selection assay for protein degraders

doi: 10.1126/sciadv.abd6263

Figure Lengend Snippet: ( A ) Immunoblot and ( B ) RT-qPCR analysis of the NCI-H1876 SCLC cell line that endogenously expresses ASCL1 infected to express the indicated sgRNAs. n = 3 biological replicates. ( C and E ) Immunoblot and ( D and F ) RT-qPCR analysis of NCI-H1876 human SCLC cells (C and D) and 97-2 mouse SCLC cells (E and F) after treatment with the CDK2 PROTAC degraders (TMX-2138 and TMX-2172) or the indicated negative controls, all used at 500 nM for either 36 hours (C and D) or 8 hours (E and F). Neg Deg, negative control degrader ZXH-7035. n = 3 biological replicates. ( G ) Immunoblot analysis and ( H ) quantification of ASCL1 protein levels in 97-2 cells first treated with the CDK2 PROTAC degrader or negative control (500 nM) for 4 hours and then treated with cycloheximide (CHX) (150 μg/ml) for the indicated times. S.E., short exposure; L.E., long exposure. n = 4 biological replicates. In all experiments, error bars represent SD except in (H), where error bars represent SEM. * P < 0.05; *** P < 0.001; **** P < 0.0001.

Article Snippet: The following compounds were purchased: POM (Selleck, #S1567), LEN (Selleck, #S1029), MG132 ( N -carbobenzyloxy- l -leucyl- l -leucyl- l -leucinal; Thermo Fisher Scientific, #47479020MG), MLN4924 (Active Biochem, #A-1139), MLN7243 (Thermo Fisher Scientific, #NC1129906), Spautin-1 (BioTechne; #5197/10), cycloheximide (VWR, #97064-724), BVdU (Chem-Impex International Inc., catalog no. 27735), actinomycin D (Thermo Fisher Scientific, #11805017), and dinaciclib (Selleck, #S2768).

Techniques: Western Blot, Quantitative RT-PCR, Infection, Negative Control