Anti-IRF5 antibody

• WB

Lab

Western blot - Anti-IRF5 antibody (AB21689)

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab21689 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

All lanes:

Western blot - Anti-IRF5 antibody (ab21689) at 1 µg/mL

Lane 1:

HeLa Whole Cell Lysate at 20 µg

Lane 2:

Mouse Liver Tissue Lysate at 10 µg

Lane 3:

Mouse Kidney Tissue Lysate at 10 µg

Lane 4:

Rat Liver Tissue Lysate at 10 µg

Secondary

All lanes:

Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/50000 dilution

Predicted band size: 56 kDa

Observed band size: 25 kDa,60 kDa,85 kDa

true

Exposure time: 8min

• WB

Project

Western blot - Anti-IRF5 antibody (AB21689)

All lanes:

Western blot - Anti-IRF5 antibody (ab21689) at 1 µg/mL

Lane 1:

HeLa whole cell lysate at 20 µg

Lane 2:

HeLa whole cell lysate at 20 µg with Human IRF5 peptide (<a href='/en-us/products/unavailable/human-irf5-peptide-ab22411'>ab22411</a>)

Secondary

All lanes:

Alexa Fluor Goat polyclonal to IgG (700) at 1/10000 dilution

Predicted band size: 56 kDa

Observed band size: 60 kDa

false

• WB

Project

Western blot - Anti-IRF5 antibody (AB21689)

All lanes:

Western blot - Anti-IRF5 antibody (ab21689) at 1 µg/mL

Lane 1:

Liver (Mouse) Tissue Lysate - normal tissue at 10 µg

Lane 2:

Kidney (Mouse) Tissue Lysate at 10 µg

Lane 3:

Liver (Rat) Tissue Lysate at 10 µg

Secondary

All lanes:

IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Predicted band size: 56 kDa

Observed band size: 60 kDa

false

• WB

CiteAb

Western blot - Anti-IRF5 antibody (AB21689)

Western Blotting using Anti-IRF5 antibody, ab21689. Publication image from Allouch, A. et al., 2022, Nat Commun, 36347876. Legend direct from paper.

Tumor cell phagocytosis triggers the proinflammatory activation of macrophages.a Schematic representation of the coculture of CMFDA-labeled MDMs with CMTMR-labeled malignant hematologic cells. b Confocal micrograph of MDMs and Jurkat cells after 8 h of coculture. c, d Percentage of phagocytosis of leukemia cells or PBLs (c) or CD34+ AML cells (d) in control (Co.)- or ZVAD (100 µM)-treated cocultures. e FACS dot plot of Phago+ MDMs or Phago- MDMs sorted after 2 h of coculture with MOLT4 cells. f, g Confocal micrographs and percentages of CMTMR+CMFDA+ MDMs at 2 h (**p = 0.0079) (f, g) and 96 h (h, i) after Phago+ MDMs and Phago- MDMs sorting. j–n Phago+ MDMs were analyzed in comparison to Phago- MDMs to characterize modulated genes by a microarray (**p = 0.0022, *p = 0.0152) (j), CD163 membrane expression by FACS (**p = 0.0039) (k), and IRF5 expression by western blot (WB) analysis (l) and the supernatant (SN) was evaluated for indicated proinflammatory cytokines by WB analysis (l) or for IFNγ by a cytokine microarray (*p = 0.0286) (m) or ELISA (***p = 0.0008) (n) at 96 h (j, k, m) and 7 d (l, n) after FACS sorting. o, p Transwell coculture model of Phago+ MDMs and Phago- MDMs at 2 h after FACS sorting (o), and iNOS expression identified by WB analysis of Phago- MDMs cocultured in the bottom chambers for 15 d (p). In b, l, p and e, f, h, the data are representative of n = 3 and n = 5 donors. In c, n, d, and g, i, the data are presented as the mean±SEM from n = 3, n = 6, and n = 5 donors. In d, the CD34+ cells were from n = 4 AML patients. In j, box plots show centre line as median, box limits as upper and lower quartiles, and whiskers as a minimum to maximum values, from n = 3 donors. In k and m, the data are donor matched from n = 9 and n = 4 donors. Exact p-values are indicated and determined with two- (g) or one-tailed (m) unpaired Mann-Whitney test, the Kolmogorov–Smirnov test (j), a two-tailed paired Wilcoxon test (k), and a two-tailed unpaired t test (n). Source data are provided as a Source Data file.

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• WB

CiteAb

Western blot - Anti-IRF5 antibody (AB21689)

Western Blotting using Anti-IRF5 antibody, ab21689. Publication image from Nishiyama, A. et al., 2014, Nat Commun, 24818655. Legend direct from paper.

IRF5 is required for fibronectin-mediated upregulation of P2X4R in microglia.(a) Representative western immunoblot of IRF5 in whole-cell lysate, and in nuclear or cytosolic components of control or IRF5 shRNA-transduced BV2 cells treated with fibronectin for 4 h. (b) Relative band density ratios of IRF5 (normalized to β-actin, lamin B orα-tublin) from control or IRF5 shRNA-transduced BV2 cells treated with fibronectin (n=4; *P<0.05, ***P<0.001). (c) Real-time PCR analysis of P2rx4 mRNA in control or Irf5 shRNA-transduced BV2 cells 6 h after fibronectin treatment. Values represent the relative ratio of P2rx4 mRNA (normalized to the value for 18s mRNA) to control shRNA-transduced cells (n=6; *P<0.05, **P<0.01, ***P<0.001). (d) Representative western immunoblot of P2X4R in control or IRF5 shRNA-transduced BV2 cells 6 h after fibronectin treatment. (e) Relative band density ratios of P2X4R (normalized to β-actin) to control shRNA-transduced cells (n=6; ***P<0.001). (f) Schematic of the four interferon-stimulated response element (ISRE) sites on the promoter region of P2X4R. (g) Chromatin immunoprecipitation (ChIP)-qPCR assay of P2rx4 promoter fragments immunoprecipitated by antibodies for IRF1, IRF3, IRF5, IRF7 or IRF9 in BV2 cells with or without fibronectin, respectively. Values represent the relative ratio of the values of BV2 cells with fibronectin (normalised to the value for normal IgG) to that of cells without fibronectin. Data are representative of three experiments. Values are the mean±s.e.m. Full-size blots are shown in Supplementary Fig. 9.

false

• WB

CiteAb

Western blot - Anti-IRF5 antibody (AB21689)

Western Blotting using Anti-IRF5 antibody, ab21689. Publication image from Nishiyama, A. et al., 2014, Nat Commun, 24818655. Legend direct from paper.

Loss of IRF5 abrogates PNI-induced tactile allodynia without affecting acute pain sensation or inflammatory pain.(a,b) PWT of Irf5−/− and WT littermates (Irf5+/+) before and after PNI (n=6; ***P<0.001 versus Pre; ##P<0.01, ###P<0.001 versus the ipsilateral side of WT mice). (c) Upper : representative western immunoblots of IRF5 in the spinal cords of mice treated with control or IRF8 siRNAs 7 days after PNI. Lower : relative band density ratios of IRF5 (normalized to β-actin) to control RNA (n=6). (d) Reversal of PNI-induced allodynia by intrathecal administration of IRF5 siRNA (20 pmol) once a day for 2 days (on day 5 and 6 after PNI) in WT mice (n=6). (e) Hot-plate test of which values represent the latencies for animals to lick their hindpaws or jump (n=6). (f) Tail-flick test of which values represent the latencies to flick their tail from the heat source (n=6). (g) Formalin test of which values indicate the duration of nociceptive behaviours (n=8). (h) Total duration (sec) of nociceptive behaviours for 0–5 min (1st phase) and for 10–60 min (2nd phase) (n=8). (i) PWT of Irf5+/+ and Irf5−/− mice before and after intraplantar CFA injection (n=4, **P<0.01, ***P<0.001 versus Pre). Values are the mean±s.e.m. Full-size blots are shown in Supplementary Fig. 12.

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