2506s western blot  (Cell Signaling Technology Inc)

Journal: EMBO Reports

Article Title: TMPRSS2-induced Golgi disruption restricts the incorporation of virus envelope glycoproteins into virions

doi: 10.1038/s44319-026-00797-2

Figure Lengend Snippet: ( A ) Phosphorylated and total ERK (pERK and ERK, respectively) were detected in 293 T cells expressing CD4-mCherry (Control), TMPRSS2-mCherry (TM2-WT), TMPRSS2-dCT-mCherry (TM2-dCT), or TMPRSS2-S441A-mCherry (TM2-S441A) at the indicated time (h) post-transfection by immunoblotting analysis. The ratio of pERK to total ERK at 0 hr was set as 1, and the ratio of pERK to total ERK at each time point to that is shown in the graph (the quantified results are expressed as mean ± SEM of three separate experiments, n = 3). The samples were obtained from the same experiment, and the gels and blots were processed in parallel. The results of Student’s t tests for the value of Ctrl at each time point are shown above the points. ( B ) IPA analysis of differentially expressed proteins between TM2-WT- and TM2-S441A-transfected 293T cells. ( C ) Experimental scheme for ( D , E ). ( D ) Inhibition of TM2-induced Golgi disruption using ERK or CDK inhibitors was analyzed in 293T cells, transfected with Golgi-YFP (green) and TM2-WT (red), and cultured for 24 h in the presence of 200 μM nafamostat. After removing nafamostat, the cells were cultured with the indicated concentrations of ERK or CDK5 inhibitor for 180 min before undergoing fixation. ( E ) Quantitative analysis of the images. Sixty cells were randomly selected from at least three independent images and plotted. Representative data from three independent experiments are shown. Scale bars indicate 50 μm. The results of Student’s t tests are shown above the bars. .

Article Snippet: CDK5 inhibitor 20-233 , MCE , HY-123772.

Techniques: Expressing, Control, Transfection, Western Blot, Inhibition, Disruption, Cell Culture

Journal: EMBO Reports

Article Title: TMPRSS2-induced Golgi disruption restricts the incorporation of virus envelope glycoproteins into virions

doi: 10.1038/s44319-026-00797-2

Figure Lengend Snippet: ( A ) Inhibition of TMPRSS2-induced Golgi disruption using ERK or CDK inhibitors, analyzed in 293T cells, transfected with Golgi-YFP (green) and TMPRSS2-WT-mCherry (TM2-WT-Cherry, red) and cultured for 24 h with 200 μM nafamostat. After removing nafamostat, cells were cultured with 20 μM ERK (20-233) or CDK1 (Ro 3306) inhibitors for 180 min before fixation. Scale bars indicate 10 μm. ( B ) Quantitative analysis of Golgi disruption in the images. Forty cells were randomly selected from at least three independent images and plotted. Representative data from two independent experiments are shown. ( C ) Immunoblotting analysis of CoV-2-S pseudotyped lentiviruses in 293T cells expressing CD4 (Ctrl), or TMPRSS2 (TM2) with or without 50 μM of ERK inhibitor. The ratio of CoV-2-S2 to p24 was calculated based on band density (right, the quantified results are expressed as mean ± SEM of three separate experiments, n = 3). ( D ) Inhibition of TM2 function by CoV-2-E. Luciferase activity for the infectivity of CoV-2-S pseudotyped lentiviruses produced in CD4-mCherry- (CD4)-, or TMPRSS2-mCherry- (TM2)-, and CoV-2-E or CoV-2-EdN14 expressing 293 T cells (the quantified results from a representative experiment in the two experiments independently conducted are expressed as mean ± SEM, n = 6). ( E ) Immunofluorescence analysis of Golgi-YFP (green) and CoV-2-E (blue) in CD4-mCherry (CD4-Cherry)-, TMPRSS2-WT-mCherry (TM2-WT-Cherry)-, TMPRSS2-dCT-mCherry (TM2-dCT-Cherry)-, or TMPRSS2-S441A-mCherry (TM2-S441A-Cherry)-expressing 293T cells cultured with CoV-2-E inhibitory peptide (+) or control peptide (−) at 24 h post-transfection. ( F ) Immunofluorescence analysis for CoV-2-S structural changes using anti-CoV-2-S2 (1A9, green) antibodies in CoV-2-S and CoV-2-E (blue) in CD4-Cherry-, TM2-WT-Cherry, TM2-dCT-Cherry, or TM2-S441A-Cherry (red) -expressing 293 T cells cultured with CoV-2-E inhibitory peptide (+) or control peptide (−) at 24 h post-transfection. Scale bars indicate 50 μm. The results of Student’s t tests are shown above the bars.

Article Snippet: CDK5 inhibitor 20-233 , MCE , HY-123772.

Techniques: Inhibition, Disruption, Transfection, Cell Culture, Western Blot, Expressing, Luciferase, Activity Assay, Infection, Produced, Immunofluorescence, Control

Journal: World Journal of Surgical Oncology

Article Title: The FGD5-AS1/miR-142-5p/CDK5 axis promotes ESCC progression by regulating mitochondrial fission and mitophagy: insights from integrative bioinformatics and experimental analyses

doi: 10.1186/s12957-026-04338-y

Figure Lengend Snippet: CDK5 as a target of miR-142-5p and its positive correlation with FGD5-AS1 expression. A qRT-PCR analysis of CDK5 expression levels in ESCC tissues and Adjacent tissue samples. ** p < 0.01; B Pearson correlation analysis of CDK5 and miR-142-5p expression levels. C - D qRT-PCR and Western blot analysis of CDK5 mRNA and protein expression levels in cells overexpressing or knocking down miR-142-5p. ** p < 0.01 vs. NC mimics; # p < 0.05, ## p < 0.01; E Predicted target sequences of CDK5 and miR-142-5p. F Dual-luciferase reporter assay validating the interaction between CDK5 and miR-142-5p. ** p < 0.01; G Pearson correlation analysis of FGD5-AS1 and CDK5 expression levels. H - I qRT-PCR and Western blot analysis of CDK5 expression levels in cells overexpressing or knocking down FGD5-AS1. * p < 0.05, ** p < 0.01

Article Snippet: After transfer, 5% non-fat milk was used to block the membranes at room temperature for 1 h. Overnight incubation at 4 °C was performed with primary antibodies against MFN1 (A21293), MFN2 (A19678), Drp1 (A2705), BECN1 (A21191), P62 (A19700), PINK1 (A25301), Parkin (A0968), CDK5 (A2689) (all 1:1000; ABclonal, China), p-Drp1 (3455), LC3-II (3868), LC3-I (4599) (all 1:1000; CST, USA), and β-actin (1:50000, AC026, ABclonal, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Luciferase, Reporter Assay

Journal: World Journal of Surgical Oncology

Article Title: The FGD5-AS1/miR-142-5p/CDK5 axis promotes ESCC progression by regulating mitochondrial fission and mitophagy: insights from integrative bioinformatics and experimental analyses

doi: 10.1186/s12957-026-04338-y

Figure Lengend Snippet: The FGD5-AS1/miR-142-5p/CDK5 axis influences ESCC progression. A Western blot analysis of CDK5 protein levels in shNC, sh-FGD5-AS1, sh-FGD5-AS1 + miR-142-5p inhibitor, and sh-FGD5-AS1 + OE-CDK5 groups of KYSE150 cells. B MTT assay of cell viability in the four groups. C TUNEL assay of apoptosis levels in the four groups. D Transwell assay of invasion capabilities in the four groups. E Scratch assay of migration capabilities in the four groups. F JC-1 assay of mitochondrial membrane potential in the four groups. G ATP assay of ATP levels in the four groups. H Western blot analysis of MFN1, MFN2, Drp1, and p-Drp1 protein levels in the four groups. I Western blot analysis of LC3-II, LC3-I, BECN1, P62, PINK1, and Parkin protein levels in the four groups. ** p < 0.01 vs. Control, ; ## p < 0.01 vs. sh-FGD5-AS1

Article Snippet: After transfer, 5% non-fat milk was used to block the membranes at room temperature for 1 h. Overnight incubation at 4 °C was performed with primary antibodies against MFN1 (A21293), MFN2 (A19678), Drp1 (A2705), BECN1 (A21191), P62 (A19700), PINK1 (A25301), Parkin (A0968), CDK5 (A2689) (all 1:1000; ABclonal, China), p-Drp1 (3455), LC3-II (3868), LC3-I (4599) (all 1:1000; CST, USA), and β-actin (1:50000, AC026, ABclonal, China).

Techniques: Western Blot, MTT Assay, TUNEL Assay, Transwell Assay, Wound Healing Assay, Migration, Membrane, ATP Assay, Control