rabbit anti p50  (Proteintech)

Journal: Gut microbes

Article Title: CD44 is a macrophage receptor for TcdB from Clostridioides difficile that via its lysine-158 succinylation contributes to inflammation.

doi: 10.1080/19490976.2025.2506192

Figure Lengend Snippet: Figure 7. Desuccinylase SUCLG2 mediated CD44 succinylation at Lys158 enhances macrophage inflammatory cytokine production via NF-κB activation. (a) Succinylation of CD44 in THP-1-Mφ, and mass spectrometric verification of CD44 succinylation at K158 (KsuccYVQKGEYR). (b) K158 (marked in red), a CD44 ortholog, is highly conserved in mammals. (c – e) expression of CD44 was measured by WB (c), production of IL-1β and IL-6 was determined by an ELISA (d), and the expression of phosphorylated p50 in the nucleus and cytoplasm was detected by WB (e) in wild-type or CD44-mutated THP-1-Mφ. (f) Results show the NF-κB-JASPAR binding motif. (g) ChIP assay coupled with qRT-PCR analysis revealed the relative enrichment of NF-κB p50 on the CD44 promoters in THP- 1-Mφ. The Fold enrichment of the ChIP assay was calculated with reference to the control IgG after normalization to the input DNA. (h) Representative western blot lines of succ-CD44Lys158 and CD44 proteins in THP-1-Mφ after treatment with TcdB/FBD. (i) Mutations in CD44 that did not disrupt the binding of THP-1-Mφ membrane extracts with TcdB or FBD were demonstrated by ELISA. (j) The protein- protein interaction (PPI) network was constructed using the STRING database based on post-translational modification data. CD44 and SUCLG2 are highlighted in red and blue, respectively. Connecting lines indicate predicted binding, interaction, or complex formation between proteins. (k) Representative western blot bands showing SUCLG2 expression in THP-1-Mφ. GAPDH served as the loading control. (l) Immunoprecipitation analysis of SUCLG2 binding to succ-CD44Lys158 in TcdB/FBD-treated macrophages. Anti-IgG served as a negative control, with GAPDH as the loading control. All data in (d) and (g – h) are shown as the mean ± SEM (n = 3). (ns: p > 0.05, *p < 0.05, **p < 0.01, &p < 0.001).

Article Snippet: The anti-phospho-NF-κB1 p105/p50 (Cat# 13586, Cell Signaling Technology) antibody was used in immunoblotting.

Techniques: Activation Assay, Expressing, Enzyme-linked Immunosorbent Assay, Binding Assay, Quantitative RT-PCR, Control, Western Blot, Membrane, Construct, Modification, Immunoprecipitation, Negative Control

Journal: PLOS Pathogens

Article Title: STAT3, MYC, and EBNA1 cooperate through a ZC3H18 transcriptional network to regulate survival and proliferation of EBV-positive lymphomas

doi: 10.1371/journal.ppat.1013166

Figure Lengend Snippet: (A, B) HH514-16 BL cells were seeded at 5 × 10 5 /ml. After 24 hours, one million cells were infected with lenti-shControl (shCtrl) or lenti-sh ZC3H18 (sh ZC3H18 #1, #2) and selected with puromycin (5 µg/mL) for 7 days followed by immunoblotting with indicated antibodies (A) or RT-qPCR to quantify IKBα , p65 , and p50 transcript levels (B). (C, D) HH514-16 cells underwent ChIP with indicated antibodies or control IgG, followed by qPCR with primers targeting the promoter regions of IKBα , p65 , and p50 (C) or gene body regions of ZC3H18 and BRCA2 (D). Data were normalized to input. (E) HH514-16 cells were seeded at 5 × 10 5 /ml. After 24 hours, one million cells were transfected with two siRNAs targeting ZC3H18 or control siRNA and harvested after another 24 hours for immunoblotting with indicated antibodies. (F) HH514-16 BL cells were seeded at 5 × 10 5 /ml. After 24 hours, one million cells were infected with lenti-shControl (shCtrl) or lenti-sh ZC3H18 (sh ZC3H18 #1, #2) in the presence of puromycin (5 µg/mL) for 7 days and then analyzed by RT-qPCR to quantify BRCA1 and BRCA2 transcripts. Error bars in B, C, D, and F represent SEM of 3 technical replicates; *, p < 0.05; **, p < 0.01; ***, p < 0.001 (versus control); NS, not significant; experiments were performed twice.

Article Snippet: The following antibodies were used for immunostaining: rabbit anti-STAT3 antibody (4904S, Cell Signaling Technology), mouse anti-EBNA1 antibody (sc-81581, Santa Cruz Biotechnology), rabbit anti-MYC antibody (A190-105A, Bethyl Laboratories), mouse anti-β-actin antibody (clone AC-15) (A1978, Sigma-Aldrich), rabbit anti-phospho-IκBα (Ser32) antibody (2859S, Cell Signaling Technology), rabbit anti-IκBα antibody (9242S, Cell Signaling Technology), rabbit anti-p65 antibody (8242S, Cell Signaling Technology), rabbit anti-p50 antibody (13586, Cell Signaling Technology), rabbit anti-ZC3H18 antibody (A304-682A, Bethyl Laboratories), rabbit anti-Caspase 3 antibody (GTX110543, GeneTex), mouse anti-Cyclin A antibody (sc-53228, Santa Cruz Biotechnology), rabbit anti-Cyclin B antibody (4138S, Cell Signaling Technology), rabbit anti-Cyclin E antibody (A301-566, Bethyl Laboratories), mouse anti-ZEBRA antibody (sc-53904, Santa Cruz Biotechnology), rabbit anti-PCNA antibody (A300-276, Bethyl Laboratories), HRP-conjugated goat anti-mouse IgG (626520, Thermo Fisher Scientific), and HRP-conjugated goat anti-rabbit IgG (31460, Thermo Fisher Scientific).

Techniques: Infection, Western Blot, Quantitative RT-PCR, Control, Transfection

Journal: World Journal of Gastroenterology

Article Title: Bicuculline ameliorates metabolic dysfunction-associated steatotic liver disease by inhibiting the nuclear factor-kappa B pathway and reducing lipid accumulation

doi: 10.3748/wjg.v31.i17.105438

Figure Lengend Snippet: Bicuculline affects lipid accumulation in free fatty acid-treated AML12 cells through the nuclear factor-kappa B pathway. A: After immunofluorescence staining, the localization of nuclear factor-kappa B p65 (green) was observed using confocal microscopy. The nuclei were stained with DAPI (blue); B and C: Representative western blotting results and density analysis of p65 and p50 expression; the results are expressed as the means ± SD ( n = 2); D: QRT-PCR analysis of Dgat1 , Dgat2 , Scd1 , Srebf1 , Fasn , IL-1β , COX2 , and TNF-α gene expression. The results are expressed as the mean ± SD ( n = 4). Compared with the model group. a P < 0.05; b P < 0.01; c P < 0.001; BIC: Bicuculline.

Article Snippet: Rabbit anti-NF-κB/p65, rabbit anti-NF-κB/p50, rabbit anti-Lamin A/C, and mouse anti-GAPDH antibodies were purchased from Proteintech (Batch Nos.

Techniques: Immunofluorescence, Staining, Confocal Microscopy, Western Blot, Expressing, Quantitative RT-PCR, Gene Expression

Journal: World Journal of Gastroenterology

Article Title: Bicuculline ameliorates metabolic dysfunction-associated steatotic liver disease by inhibiting the nuclear factor-kappa B pathway and reducing lipid accumulation

doi: 10.3748/wjg.v31.i17.105438

Figure Lengend Snippet: Bicuculline affects lipid accumulation in metabolic dysfunction-associated steatotic liver disease model mice through the nuclear factor-kappa B pathway. A: Representative images of immunohistochemical staining for p65 (brown); B and C: Representative western blotting results and density analysis of p50 and p65 expression; the results are expressed as the means ± SD ( n = 3); D: QRT-PCR analysis of Dgat1 , Dgat2 , Scd1 , Srebf1 , Fasn, IL-1β , COX2 , and TNF-α gene expression. The results are expressed as the mean ± SD ( n = 4). Compared with the model group. a P < 0.05; b P < 0.01; c P < 0.001; BIC: Bicuculline.

Article Snippet: Rabbit anti-NF-κB/p65, rabbit anti-NF-κB/p50, rabbit anti-Lamin A/C, and mouse anti-GAPDH antibodies were purchased from Proteintech (Batch Nos.

Techniques: Immunohistochemical staining, Staining, Western Blot, Expressing, Quantitative RT-PCR, Gene Expression