mouse gapdh  (Cell Signaling Technology Inc)

Journal: Scientific Reports

Article Title: RNase 7 and Th cytokines synergistically increase the secretion of interleukin-6 from keratinocytes

doi: 10.1038/s41598-025-04403-8

Figure Lengend Snippet: IL-24 secretion correlates with and depends on IL-6 secretion and signaling and IL-6 secreted from R7-stimulated cells activates STAT3 signaling. ( a ) We calculated the ratio of cytokine release from unstimulated and R7-stimulated HPK and plotted the IL-24 ratio against the IL-6 ratio. Statistics: Pearson correlation, significant with p = 0.004, n = 15. ( b ) HPK were starved for 4 h and then stimulated for 15 min with either 10 ng/mL rIL-6, 10 ng/mL Hyper-IL-6 (Hy-IL-6) or left unstimulated. Phosphorylation of STAT3 and total STAT3 protein levels were visualized by immunoblot in comparison to housekeeping GAPDH protein. ( c ) pSTAT3/STAT3 ratios of four HPK donors were quantified and statistical significance was analysed by paired t test; **p < 0.01. Bars indicate median. ( d , e ) HPK of 9 donors were seeded on a 24-well plate and were not stimulated (ctr) or stimulated with 5 µg/ml R7 either in the absence or in the presence of 2 µg/ml of a function-blocking antibody against IL-6. After 72 h, we measured IL-24 transcript levels relative to GAPDH transcripts (d) and IL-24 release by ELISA ( e ). Data in ( d , e ) was statistically analyzed by Friedman’s test followed by Dunn’s multiple comparisons test. *p < 0.05, **p < 0.01, ns, not significant. Bars indicate median. Relative IL6R ( f ) and IL6ST ( g ) expression of HPK (n = 6) after 48 h without (ctr) or with 5 µg/ml R7 was determined by qRT-PCR and compared by Wilcoxon matched-pairs signed rank ( f ) or paired t test ( g ). Bars indicate median. ( h ) HPK were starved for 4 h and then stimulated for 15 min either directly with 5 µg/ml R7 or 10 ng/ml rIL-6 or with spnts of HPK that had been stimulated for 48 h with 5 µg/ml R7 or left unstimulated, followed by either no treatment or an incubation with 2 µg/ml of a function-blocking IL-6 antibody or an isotype control antibody for 1 h at 37°C. Phosphorylation of STAT3 was analysed by immuno-blotting ( i ) Quantification of pSTAT3 relative to STAT3 bands (n = 5). Statistical analysis was performed with Friedman’s test followed by Dunn’s multiple comparisons test. *p < 0.05. Bars represent median.

Article Snippet: Proteins were probed using monoclonal rabbit anti-pSTAT3 antibody (pY705, D3 A7; cat. #9145), monoclonal mouse anti-STAT3 antibody (124H6; cat. #9139) or monoclonal mouse anti-GAPDH antibody (cat. #14 C10; Cell Signaling Technology, MA, USA) at 1:1000 dilution and labeled with secondary anti-rabbit IgG HRP-linked antibody (cat. #7074; Cell Signaling Technology, MA, USA) or anti-mouse IgG HRP-linked antibody (cat. #7076; Cell Signaling Technology, MA, USA) at 1:2000 dilution, respectively.

Techniques: Phospho-proteomics, Western Blot, Comparison, Blocking Assay, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Incubation, Control

Journal: International Journal of Molecular Sciences

Article Title: Involvement of lncRNAs NEAT1 and ZBTB11-AS1 in Active and Persistent HIV-1 Infection in C20 Human Microglial Cell Line

doi: 10.3390/ijms26104745

Figure Lengend Snippet: HIV-1 initially replicates actively in microglia and persists with low RNA and viral protein expression. C20 cells were infected with VSVg-pseudotyped HIV-1. Non-infected cells were used as a control (mock). ( A ) At 1, 4, and 21 days p.i., cell extracts were used to extract total RNA, and intracellular genomic RNA (gRNA) and GAPDH were quantified using RT-qPCR. gRNA was normalized to day 1 p.i. Data were analyzed using Student’s t -test, n = 3, ns ≥ 0.05, * p < 0.05, **** p < 0.0001. ( B ) In parallel, cell extracts were used to detect anti-CAp24 using Western blotting. A representative figure from one of the three experiments ( n = 3) is shown, with GAPDH as a loading control. ( C ) gRNA in the supernatant (sup) was detected using RT-qPCR. Copies/mL of cDNA obtained from gRNA in supernatants were measured on days 1 and 4 p.i. using the number of DNA copies with the length of the plasmid and DNA concentration (ng/µL). Data were analyzed t-student test, n = 3, ns ≥ 0.05. ( D ) DNA was extracted from cells in all conditions. HIV-1 DNA integrated into the host genome was detected on days 1, 4, and 21 p.i. using PCR with ALU-Gag primers and then R-U5 primers, n = 3 .

Article Snippet: GAPDH was evaluated as a protein loading control using a GAPDH mouse mAb (HRP-conjugated) antibody (1:1000 dilution) (Cell Signaling, #51332S).

Techniques: Expressing, Infection, Control, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Involvement of lncRNAs NEAT1 and ZBTB11-AS1 in Active and Persistent HIV-1 Infection in C20 Human Microglial Cell Line

doi: 10.3390/ijms26104745

Figure Lengend Snippet: NEAT1 changes its expression and subcellular localization during active and persistent HIV-1 replication in microglia. C20 cells were infected with VSVg-pseudotyped HIV-1. Non-infected cells were used as a control (mock). At 4 and 21 days p.i., cell extracts were used to extract total, cytosolic, and nuclear RNA. The expression level of NEAT1 was detected using RT-qPCR, and GAPDH was detected as a reference gene. Data were analyzed using Student’s t -test, n = 3, * p < 0.05, ** p < 0.01. *** p < 0.001. ( A ) NEAT1 in total RNA on day 4 p.i. ( B ) NEAT1 in total RNA on day 21 p.i. ( C ) NEAT1 in cytosolic RNA on day 4 p.i. ( D ) NEAT1 in cytosolic RNA on day 21 p.i. ( E ) NEAT1 in nuclear RNA on day 4 p.i. ( F ) NEAT1 in nuclear RNA on day 21 p.i.

Article Snippet: GAPDH was evaluated as a protein loading control using a GAPDH mouse mAb (HRP-conjugated) antibody (1:1000 dilution) (Cell Signaling, #51332S).

Techniques: Expressing, Infection, Control, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Involvement of lncRNAs NEAT1 and ZBTB11-AS1 in Active and Persistent HIV-1 Infection in C20 Human Microglial Cell Line

doi: 10.3390/ijms26104745

Figure Lengend Snippet: ZBTB11-AS1 changes its expression and subcellular localization during active and persistent HIV-1 replication in C20 microglial cells infected with VSVg-pseudotyped HIV-1. Non-infected cells were used as a control (mock). At 1, 4, and 21 days p.i., cell extracts were used to extract total, cytosolic, and nuclear RNA. The expression level of ZBTB11-AS1 was detected using RT-qPCR, and GAPDH was detected as a reference gene. Data were analyzed using Student’s t -test, n = 3, ns ≥ 0.05, ** p < 0.01, *** p < 0.001. ( A ) ZBTB11-AS1 in total RNA on day 4 p.i. ( B ) ZBTB11-AS1 in total RNA on day 21 p.i. ( C ) ZBTB11-AS1 in cytosolic RNA on day 4 p.i. ( D ) ZBTB11-AS1 in cytosolic RNA on day 21 p.i. ( E ) ZBTB11-AS1 in nuclear RNA on day 4 p.i. ( F ) ZBTB11-AS1 in nuclear RNA on day 21 p.i.

Article Snippet: GAPDH was evaluated as a protein loading control using a GAPDH mouse mAb (HRP-conjugated) antibody (1:1000 dilution) (Cell Signaling, #51332S).

Techniques: Expressing, Infection, Control, Quantitative RT-PCR

Journal: bioRxiv

Article Title: A genus-wide interaction atlas across NS4B orthologues identifies a conserved role for UFMylation in orthoflavivirus replication

doi: 10.1101/2025.05.15.653649

Figure Lengend Snippet: a. Schematic representation of the NS4B protein membrane topology depicting the position of the internal HA tag. b. Following infection of JEG-3 cells with either ZIKV H/PF/2013 wild-type strain (ZIKV, black) or ZIKV-NS4B HA (blue) at an MOI 0.01, supernatants were collected at the indicated time points to determine the kinetics of infectious virus release by TCID50 (Tissue Culture Infectious Dose) assay. c. Western blot analysis confirmed the expression of HA tagged NS4B protein during ZIKV infection (top and middle panels, third lane). GAPDH was used as a loading control (bottom panel). The cell lysates were harvest in RIPA buffer 48 hpi. d. Representative images of JEG-3 cells infected with replication-competent ZIKV H/PF/2013 molecular clone carrying an HA-tag within the NS4B ORF (ZIKV-NS4B HA ) at an MOI of 5. The cells were fixed 24 hpi, stained with anti-NS4B (green) and anti-HA (red) antibodies, and visualized by confocal microscopy. Scale bar 20 µm. Panel b, n=3 biological replicates are shown, each circle represents the mean and error bars represent the standard deviation of the mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, or ns, not significant as determined by unpaired two-tailed t-test on log₁₀-transformed PFU/mL values with Holm–Šidák correction for multiple comparisons. Panel c shows representative immunoblot.

Article Snippet: The mouse monoclonal antibodies recognizing HA (2999), the rabbit monoclonal anti-human COXIV (4850) antibody, and HRP-conjugated anti-human GAPDH (51332) were purchased from Cell Signaling.

Techniques: Membrane, Infection, Virus, Western Blot, Expressing, Control, Staining, Confocal Microscopy, Standard Deviation, Two Tailed Test, Transformation Assay

Journal: bioRxiv

Article Title: A genus-wide interaction atlas across NS4B orthologues identifies a conserved role for UFMylation in orthoflavivirus replication

doi: 10.1101/2025.05.15.653649

Figure Lengend Snippet: a. Schematic representation of the UFMylation pathway inhibition by the UBA5 inhibitor DKM 2-93. DKM 2-93 competitively binds to the catalytic cysteine of UBA5, and prevents the activation of UFM1. b. Dose-response curve of DKM 2-93 for inhibition of ZIKV infection in JEG-3 cells. JEG-3 cells were treated with increasing concentrations of the inhibitor for 24 h, then infected with the ZIKV H/PF/2013 reporter strain expressing Renilla luciferase (MOI 0.1), and treated with the inhibitor for another 24 h. Cell viability and virus replication were determined at 24 hpi by resazurin and luciferase assays, respectively. c. Western blot analysis confirmed the impairment of virus replication. NS1 was stained as an infection marker (top panel), and GAPDH was used as a loading control (bottom panel). d. A reduction in ZIKV titers was observed in the presence of non-cytotoxic concentrations of the inhibitor in JEG-3 cells. JEG-3 cells were infected with ZIKV H/PF/2013 wild-type strain (MOI 0.01), and treated with either DMSO or two non-cytotoxic concentrations of DKM 2-93. Supernatants were harvested at 48 hpi and the virus titers were measured by plaque assay. e. Time-of-addition analysis of the antiviral activity of DKM 2-93. JEG-3 cells were either pre-treated (grey bar) with the inhibitor (32 µM) for 3 h prior to infection with ZIKV H/PF/2013 wild-type strain (MOI 0.01), or co-treated (blue bar) by adding the inhibitor (32 µM) to the viral inoculum during 1 h of virus adsorption, or post-treated (orange bar) 3 h after the removal of the viral inoculum. In each case, the supernatant was collected at 48 hpi, and the virus titers were determined by plaque assay. f-g. Huh7 cells were electroporated with wild-type subgenomic replicon (sgZIKV) reporter virus RNA expressing Renilla luciferase, and treated with DKM 2-93 (32 µM) immediately thereafter. Luciferase activity was measured 4 hours post electroporation to assess effects on viral RNA translation (f, sgZIKV-R2A), and up to 96 hours post electroporation to assess effects of viral RNA replication (g, sgZIKV-R2A). Panels d, e, and f, n=3 biological replicates; bars represent the mean and error bars represent the standard deviation of the mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, or ns, not significant as determined by one-way ANOVA with Dunnett’s multiple comparisons test (d) or two-way ANOVA with Sidak’s multiple comparisons test (e and f) or unpaired two-tailed t-test on log₁₀-transformed PFU/mL values with Holm–Šidák correction for multiple comparisons (g). Panel c, representative immunoblots from n=3 biological replicates are shown.

Article Snippet: The mouse monoclonal antibodies recognizing HA (2999), the rabbit monoclonal anti-human COXIV (4850) antibody, and HRP-conjugated anti-human GAPDH (51332) were purchased from Cell Signaling.

Techniques: Inhibition, Activation Assay, Infection, Expressing, Luciferase, Virus, Western Blot, Staining, Marker, Control, Plaque Assay, Activity Assay, Adsorption, Electroporation, Standard Deviation, Two Tailed Test, Transformation Assay