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: The Journal of Biological Chemistry

Article Title: The ATPase ATP6V1A facilitates rabies virus replication by promoting virion uncoating and interacting with the viral matrix protein

doi: 10.1074/jbc.RA120.014190

Figure Lengend Snippet: Transfection of ATP6V1A or its truncate trans -complements decreased ERA uncoating in ATP6V1A depleted cells. HEK293T cells were transfected with ATP6V1A siRNA or scrambled siRNA. At 48 h post transfection of siRNA, cells were transfected with the indicated plasmids for 24 h and infected with ERA at an MOI of 100 for 1 h on ice. Cells were then treated with PBS (pH 5) for 15 min, washed, and incubated at 37 °C for 2 h in the presence of cycloheximide. RABV M protein ( green ) and cell nuclei ( blue ) were stained and observed for M protein aggregation by confocal microscopy. Yellow solid arrows indicate high-intensity punctate staining of M proteins ( A ). Quantitative analysis of RABV uncoating in virus-infected cells. On the basis of the confocal microscopy in A , the infected cells at 3 h post infection was categorized into two types, containing more than 10 high-intensity punctate staining spots in each cell, or containing less than 10 spots in each cell. The results shown were calculated from 130 cells under a confocal microscope with a 20× objective lens ( B ).

Article Snippet: At 48 h post transfection, the cells were incubated with 5 μM protein synthesis inhibitor cycloheximide (CHX; APExBIO, USA) for 1 h at 37 °C and then incubated with ERA virus at an MOI of 100 for 1 h at 37 °C in the presence of CHX.

Techniques: Transfection, Infection, Incubation, Staining, Confocal Microscopy, Virus, Microscopy

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: Nature Communications

Article Title: Microbiota-reprogrammed phosphatidylcholine inactivates cytotoxic CD8 T cells through UFMylation via exosomal SerpinB9 in multiple myeloma

doi: 10.1038/s41467-025-57966-5

Figure Lengend Snippet: a Representative image of Co-IP using a UFM1 antibody in CD8 T cells. Rabbit IgG was used as a negative control. Mean± SD, n = 3 biological replicates, data represent two independent experiments. b TP53 protein level in CD8 T cells transfected with or without UFM1 shRNAs. Mean± SD, n = 3 biological replicates, data represent two independent experiments. c TP53 protein level detected in CD8 T cells transfected with or without UFM1 shRNAs after cycloheximide treatment. Mean± SD, n = 3 biological replicates, data represent two independent experiments. d UFM1 protein level in CD8 T cells transfected with or without the Sb9 expression vector. e Representative images of Co-IP using TP53 antibody in scrambled and Sb9 expression vector (0.5, 1, and 2 μg)-transfected CD8 T cells. Rabbit IgG was used as a negative control. Mean± SD, n = 3 biological replicates, data represent two independent experiments. f TP53 protein level in CD8 T cells transfected with or without UFC1 shRNAs. Mean± SD, n = 3 biological replicates, data represent two independent experiments. g Representative image of Co-IP using a UFM1 antibody in CD8 T cells. Mean± SD, n = 3 biological replicates, data represent two independent experiments. Rabbit IgG was used as a negative control. Sb9 SerpinB9, NC negative control, OE overexpression, UFC1, ubiquitin-fold modifier conjugating enzyme 1. All statistical tests were two-sided. Statistical significance was assessed using one-way ANOVA followed by post-hoc Tukey’s test for ( b , c , e , and f ), which was performed utilizing unpaired Student’s t test for a , d , and g . Adjustments were made for multiple comparisons. * P < 0.05; ** P < 0.01; *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: When grown to ~80% confluence, CD8 T cells were treated with 50 μg/mL cycloheximide (IC0720, Solarbio) for 3 h, 6 h, and 12 h. Then, CD8 T cells were collected, followed by the detection of TP53 levels using WB.

Techniques: Co-Immunoprecipitation Assay, Negative Control, Transfection, Expressing, Plasmid Preparation, Over Expression, Ubiquitin Proteomics

Journal: Antioxidants

Article Title: Bardoxolone-Methyl (CDDO-Me) Suppresses Androgen Receptor and Its Splice-Variant AR-V7 and Enhances Efficacy of Enzalutamide in Prostate Cancer Cells

doi: 10.3390/antiox9010068

Figure Lengend Snippet: Transcriptional and post translational regulation of AR by CDDO-Me. ( A ) 22Rv1 cells were pretreated (2 h) with 5 μg/mL cycloheximide (CHX) followed by exposure to CDDO-Me (500 nM) for 0–24 h and AR levels were monitored by western immunoblot. A representative immunoblot of AR and GAPDH protein levels in 22Rv1 cells. ( B ) The normalized data are expressed as fold changes (mean ± SEM) in two independent experiments and significant differences between groups are shown as * p < 0.05. ( C,D ) 22Rv1 cells were treated with CDDO-Me (500 nM), total RNA extracted after 3, 6, and 9 h and quantitative RT-PCR (qRT-PCR) was performed. The normalized fold change in ( C ) AR-FL and ( D ) AR-V7 gene expression from two independent experiments is expressed as the mean ± SEM. Significant differences between groups are shown as p -values (* p < 0.05; ** p < 0.005).

Article Snippet: Cycloheximide (CHX) was bought from Cayman chemicals (Ann Arbor, MI, USA).

Techniques: Western Blot, Quantitative RT-PCR, Gene Expression