loading control β tubulin  (Proteintech)

Journal: PLOS One

Article Title: Exosomal miR-320b regulates cardiomyocyte FOXM1 expression and may serve as an early-stage compensatory mechanism in obstructive sleep apnea

doi: 10.1371/journal.pone.0332862

Figure Lengend Snippet: (a) Representative transmission electron microscopy (TEM) images of exosomes isolated from plasma samples. The images depict the cup-shaped morphology of Ctrl -exo (left panel) and OSA-exo (right panel). Scale bars = 100 nm. (b) WB analysis showing the presence of the exosomal markers TSG101 and CD9 and the absence of Calnexin, a common exosomal contaminant, indicating high purity of the exosomes. HEK293T cell lysate was used as a control to verify the specificity of exosomal markers and rule out contamination. (c) Nano-flow cytometry (nFCM) analysis showing the presence of the exosome surface markers CD9 and CD81 and the absence of IgG, a negative control, in both Ctrl-exo (upper panel) and OSA-exo (lower panel). (d) Particle size distribution of exosomes as determined by nFCM. Both Ctrl-exo (left panel) and OSA-exo (right panel) displayed a typical size range of 30–150 nm.

Article Snippet: After blocking in 5% non-fat dry milk in TBST for 30 min, the membranes were incubated overnight at 4°C with primary antibodies (1:1000, Abcam) toward TSG101 (ab125011), CD9 (BOSTER, BM4212), Calnexin (ab22595), FOXM1 (ab207298), and β-Tubulin (Sinobiological, 100109-MM05T), respectively.

Techniques: Transmission Assay, Electron Microscopy, Isolation, Clinical Proteomics, Control, Flow Cytometry, Negative Control

Journal: PLOS One

Article Title: Exosomal miR-320b regulates cardiomyocyte FOXM1 expression and may serve as an early-stage compensatory mechanism in obstructive sleep apnea

doi: 10.1371/journal.pone.0332862

Figure Lengend Snippet: (a–d) mRNA expression levels of FOXM1 (a), STRA6 (b), NPY1R (c), and MSANTD1 (d) in AC16 cardiomyocytes treated with Ctrl-exo or OSA-exo for 48 hours, determined by RT-qPCR. Data are presented as mean ± SD. ****P < 0.0001. (e) RT-qPCR analysis of hsa-miR-320b levels in Ctrl-exo and OSA-exo. *P = 0.0285, n = 6 per group. (f) Schematic representation of the FOXM1 3′-UTR luciferase reporter assay. The wild-type (WT) and mutant (MUT) 3′-UTR sequences of FOXM1 are shown, highlighting the predicted hsa-miR-320b binding site. (g) RT-qPCR analysis of hsa-miR-320b levels in HEK293T cells co-transfected with hsa-miR-320b or miR-NC and the psiCHECK-2 luciferase reporter plasmid, confirming successful transfection. Data are presented as mean ± SD. ****P < 0.0001. (h) Luciferase activity in HEK293T cells normalized to Renilla luciferase, showing that co-transfection of hsa-miR-320b significantly reduced luciferase activity in cells carrying the WT FOXM1 3′-UTR construct, while no significant change was observed in those carrying the MUT 3′-UTR construct. Data are presented as mean ± SD. ****P < 0.0001, ns = not significant. (i) Hsa-miR-320b and FOXM1 mRNA levels in AC16 cardiomyocytes transfected with hsa-miR-320b mimic, inhibitor, or their respective controls (miR-NC, inhibitor-NC) for 48 hours, determined by RT-qPCR. Data are presented as mean ± SD. ****P < 0.0001, ***P < 0.001, *P = 0.0168. (j) WB analysis of FOXM1 protein levels in AC16 cardiomyocytes transfected with hsa-miR-320b mimic, inhibitor, or their respective controls (miR-NC, inhibitor-NC) for 48 hours. β-Tubulin served as a loading control. The bar graph shows quantified FOXM1 protein levels normalized to β-Tubulin. Data are presented as mean ± SD. ***P < 0.001, *P = 0.0311, n = 3.

Article Snippet: After blocking in 5% non-fat dry milk in TBST for 30 min, the membranes were incubated overnight at 4°C with primary antibodies (1:1000, Abcam) toward TSG101 (ab125011), CD9 (BOSTER, BM4212), Calnexin (ab22595), FOXM1 (ab207298), and β-Tubulin (Sinobiological, 100109-MM05T), respectively.

Techniques: Expressing, Quantitative RT-PCR, Luciferase, Reporter Assay, Mutagenesis, Binding Assay, Transfection, Plasmid Preparation, Activity Assay, Cotransfection, Construct, Control

Journal: bioRxiv

Article Title: A Suite of Foundation Models Captures the Contextual Interplay Between Codons

doi: 10.1101/2024.10.10.617568

Figure Lengend Snippet: a) We use different scoring fashions for pathogenicity of synonymous variants. Specifically, we report log likelihood ratio between wild-type and mutated codons for EnCodon and log ratio of wild-type and mutated sequence likelihoods for DeCodon models. To nominate variants for experimental validation, We fetched all synonymous variants from ClinVar and COSMIC Census Mutations databases and computed pathogenicity scores for all of our pre-trained EnCodons and DeCodons. Next, we selected synonymous variants with extremely pathogenic scores (above 99th quantile of the distribution). b) Experimental measurement of gene expression levels for AGPAT2 and RAC2 are shown for wild-type vs. mutated coding sequence. Each dot denotes an individual replicates and bars show the average expression across replicates. c) Immunoblots of FLAG-tagged AGPAT2 and RAC2 protein variants expressed in HEK293T cells, showing three biological replicates for both wild-type (Ref) and mutated (Mut) sequences. β -tubulin signal is used as a loading control. d) Geneset Enrichment Analysis of top 20 abundant genes with highest number of “pathogenic” variants with extreme scores. d) Lollipop plot of extreme variants predicted for BCR and MYC are shown across protein sequence annotated by functional domains extracted from InterPro.

Article Snippet: After blocking the membrane in SuperBlock blocking buffer (Thermo Fisher Scientific, #37537) for 10 min at room temperature, it was incubated overnight at 4°C with rabbit anti-FLAG monoclonal antibody (Cell Signaling, #14793) and mouse anti- β -tubulin monoclonal antibody (Sino Biological, #100109-MM05T), both diluted 1:2,000 in blocking buffer.

Techniques: Sequencing, Expressing, Western Blot, Control, Functional Assay

Journal: bioRxiv

Article Title: A Suite of Foundation Models Captures the Contextual Interplay Between Codons

doi: 10.1101/2024.10.10.617568

Figure Lengend Snippet: a) Barplot of observed gene expression levels of 4 other tested synonymous variants (2 controls and 2 predicted as pathogenic). b) Immunoblots of FLAG-tagged RRAS and YWHAG protein variants expressed in HEK293T cells, showing three biological replicates for both wild-type (Ref) and mutated (Mut) sequences. β -tubulin signal is used as a loading control. Lollipop plots showing potential synonymous variants with extremely pathogenic score for c) SMARCA4, d) RET, e) STK11, and f) SRC.

Article Snippet: After blocking the membrane in SuperBlock blocking buffer (Thermo Fisher Scientific, #37537) for 10 min at room temperature, it was incubated overnight at 4°C with rabbit anti-FLAG monoclonal antibody (Cell Signaling, #14793) and mouse anti- β -tubulin monoclonal antibody (Sino Biological, #100109-MM05T), both diluted 1:2,000 in blocking buffer.

Techniques: Expressing, Western Blot, Control

Journal: bioRxiv

Article Title: Allosteric degraders induce CRL5 ASB8 mediated degradation of XPO1

doi: 10.1101/2024.10.07.617049

Figure Lengend Snippet: a , XPO1 levels in HT1080 cells treated with KPT-185, LMB or DMSO detected by Western blot. b , HT1080 cells, treated with DMSO or KPT-185 for 24 h followed by fresh media with or without KPT-185, were collected at 24, 48 and 72 h for detection of XPO1 levels by Western blot (WB). c, HT1080 cells treated with DMSO or selinexor for 2 h followed by MG132 or MLN4924 for the next 10 h and endogenous XPO1 detected by WB. d, HT1080 cells transfected with RFP XPO1 WT or FLAG XPO1 C528T were treated with DMSO or KPT-185; XPO1 detected by WB. Loading control in a-d is β-tubulin. e, HT1080 cells treated with siRNA for several cullins followed by treatment with DMSO or KPT-185. Loading control is β-actin.

Article Snippet: Immunoblotting was used to detect XPO1 (CRM1 rabbit polyclonal H-300; sc-5595, Santa Cruz or CRM1 mouse monoclonal (C-1): sc-74454, Santa Cruz) and β-actin or β-tubulin (Loading Control Antibody Sampler Kit #5142, Cell Signaling Technology) to confirm equal protein loading.

Techniques: Western Blot, Transfection, Control

Journal: bioRxiv

Article Title: Allosteric degraders induce CRL5 ASB8 mediated degradation of XPO1

doi: 10.1101/2024.10.07.617049

Figure Lengend Snippet: a, HEK293T cells were transfected with XPO1 WT, XPO1 EH or XPO1 h10/11 alone or together with ASB8 WT and treated overnight with selinexor. XPO1 degradation was assessed by WB. b, Transfection with ASB8 WT, ASB8 ANK3/4 and ASB8 mutants R197G/L199G, R197G/L199R or G198R/L199R followed by overnight treatment with selinexor and WB analysis of XPO1. Representative WB images are shown in a and b with β-tubulin as a loading control. For densitometric analysis, normalized XPO1/β-tubulin peak area values from three separate experiments were averaged. c , Cell viability assay on HAP1 cells with stable overexpression of ASB8 WT or one of the ASB8 mutants. Data points were normalized to untreated cells and represent mean ±SD of three separate experiments.

Article Snippet: Immunoblotting was used to detect XPO1 (CRM1 rabbit polyclonal H-300; sc-5595, Santa Cruz or CRM1 mouse monoclonal (C-1): sc-74454, Santa Cruz) and β-actin or β-tubulin (Loading Control Antibody Sampler Kit #5142, Cell Signaling Technology) to confirm equal protein loading.

Techniques: Transfection, Control, Viability Assay, Over Expression