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

Journal: Cells

Article Title: Dynamics of T-Cell Intracellular Antigen 1-Dependent Stress Granules in Proteostasis and Welander Distal Myopathy under Oxidative Stress

doi: 10.3390/cells11050884

Figure Lengend Snippet: TIA1a WT/WDM expression does not alter phosphorylation/dephosphorylation dynamics in an oxidative stress model. ( A , B ) Western blot of total cell extracts obtained from FT293 GFP-TIA1a WT ( A ) and FT293 GFP-TIA1a WDM ( B ) lines subjected to different treatments indicated in the legend at the top. Analyses were performed with anti-TIA1, anti-eIF2α-phosphorylated (P), anti-eIF2α (Total), anti-HuR, and anti-tubulin-α (TUBA) antibodies from three independent experiments. Molecular weight markers (kDa) and identification of each band are shown. The + and − signs indicate whether the sample contains the reagent (Tet, NaAsO 2 , CHX) or not. The +/− sign indicates that NaAsO 2 is added and removed after 1 h. Abbreviations: Tet, tetracy-cline; NaAsO 2 , sodium arsenite; CHX, cycloheximide.

Article Snippet: Oxidative stress was induced as above, and cycloheximide (CHX; 5 μg/mL; Merck, Darmstadt, Germany) was also used, as a control.

Techniques: Expressing, Phospho-proteomics, De-Phosphorylation Assay, Western Blot, Molecular Weight

Journal: Journal of Cancer Prevention

Article Title: Overcoming the Intrinsic Gefitinib-resistance via Downregulation of AXL in Non-small Cell Lung Cancer

doi: 10.15430/JCP.2019.24.4.217

Figure Lengend Snippet: Enhancement of AXL degradation by yuanhuadine (YD) in H1299 cells. (A, C) Cells were treated with 25 μg/mL cycloheximide (CHX) and 10 nM YD for the indicated times. Cell lysates were analyzed by Western blot with an antibody against AXL. β-actin was used as a loading control. (B, D) AXL expression levels were quantifies by densitometry using ImageJ.

Article Snippet: Cycloheximide (CHX) was purchased from A.G. Scientific (San Diego, CA, USA).

Techniques: Western Blot, Control, Expressing

Journal: Molecular Oncology

Article Title: Endothelin‐converting enzyme‐1c promotes stem cell traits and aggressiveness in colorectal cancer cells

doi: 10.1002/1878-0261.12609

Figure Lengend Snippet: Mutation of Lys‐6 to Arg enhances stability of ECE1c. (A) DLD‐1 clone cells expressing Flag‐tagged ECE1c WT or ECE1c K6R proteins were incubated with 20 μg·mL −1 cycloheximide (CHX) in the absence (for 36 h) or presence (for 12 h) of 25 μ m silmitasertib. ECE1c proteins were detected by western blot with an anti‐Flag antibody, using β‐actin as loading control. Representative blots are shown (upper). Relative levels (%) of Flag‐ECE1c proteins from three independent experiments were calculated (lower). (B) DLD‐1 clones described in A were grown in medium without FBS for 48 h. Culture supernatants were used to measure ET‐1 levels using ELISA according to manufacturer’s instructions (Thermo Fisher). Data represent average ± SEM ( n = 3). ANOVA and Tukey tests were used. * P ≤ 0.05, ** P ≤ 0.01.

Article Snippet: Cells (5 × 10 5 ) were seeded into P60 plates and cultured for 36 h in complete medium under normal conditions, with 20 μg·mL −1 cycloheximide (CHX) in the absence or presence of 25 μ m silmitasertib (ApexBio Technology LLC, Houston, TX, USA).

Techniques: Mutagenesis, Expressing, Incubation, Western Blot, Clone Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Functional and structural deficiencies of Gemin5 variants associated with neurological disease

doi: 10.1101/2022.01.25.477707

Figure Lengend Snippet: (A) HEK293 cells expressing the wild type version of G5 845-1508 , side by side to the variants R1016C, D1019E or L1367P during 12 h were treated (+) or not (-) with cycloheximide (CHX) for additional 16 h. Samples were taken at 0, 12 and 16 h post CHX treatment. The intensity of each protein at the indicated time was determined by WB. A long exposure is shown for L1367P protein. (B) Steady-state analysis of Xpress-G5 845-1508 mRNA levels present in transfected cells at the time of harvesting determined by RTqPCR. (C) Values represent the protein intensity (mean ± SEM) of three independent experiments relatively to time 0. Asterisks denote P -values (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: For cycloheximide (CHX) chase experiments, CHX (100 μg/ml) (Merck) was added to stop translation at 12 h or 24 h post-transfection (hpt) for Xpress-G5 845-1508 or Xpress-Gemin5 proteins, respectively.

Techniques: Expressing, Transfection