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: 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

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: Drug-induced eRF1 degradation promotes readthrough and reveals a new branch of ribosome quality control

doi: 10.1101/2023.01.31.526456

Figure Lengend Snippet: (A) Immunoblot showing the protein levels of GCN1 and eRF1 in HEKR4 PTC reporter cells treated with negative control (Ctrl) or GCN1-targeting siRNA (GCN1 KD) in combination with a 6-hour incubation of 2.5 μM NVS1.1 or DMSO as control treatment. Vinculin served as a loading control. (B) Representative immunoblot showing protein levels of eRF1 and GAPDH (loading control) of HEKR4 PTC reporter cells treated with 2.5 μM NVS1.1 for 6 hours in the absence or presence of the translation elongation inhibitor cycloheximide (CHX). (C) (Top) Polysome profile showing A 260 readout of lysate deriving from HEKR4 PTC reporter cells treated with 25 μM NVS1.1 for 30 min that was separated over a 15%-50% sucrose gradient and fractionated. (Bottom) The proteins in every odd-numbered fraction of the gradient were precipitated and analyzed by immunoblotting for the indicated proteins. The fractions 3 and 5 were diluted 1/15 and 1/3, respectively. (D). Heavy polysome fractions from (C) were pooled and the proteins were analyzed by label-free mass spectrometry. Detected diGly events on lysine residues indicative of ubiquitination were normalized to the corresponding protein abundance. The fold change of the diGly frequency (log 2 diGly (NVS1.1/DMSO)) is shown for the protein eRF1 and for ribosomal proteins that showed a statistically significant difference (pVal < 0.05) upon NVS1.1 treatment

Article Snippet: The NVS compounds, Bortezomib (BTZ, Merck, Cat# 504314), MLN4924 (Selleckchem, Cat# S7109) and Cycloheximide (CHX, Focus Biomolecules, Cat# 10-117) were all dissolved in DMSO, which also served as vehicle control.

Techniques: Western Blot, Negative Control, Incubation, Control, Mass Spectrometry, Ubiquitin Proteomics, Quantitative Proteomics

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