Mouse ICAM-1/CD54 Antibody

ICAM‑1/CD54 in Mouse Testis.

ICAM-1/CD54 was detected in perfusion fixed frozen sections of mouse testis using Goat Anti-Mouse ICAM-1/CD54 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF796) at 15 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008) and counterstained with hematoxylin (blue). Specific staining was localized to plasma membrane in Leydig cells. View our protocol for Chromogenic IHC Staining of Frozen Tissue Sections.

Detection of Mouse ICAM-1/CD54 by Western Blot

Increased stress kinase signaling and JNK pathway-dependent cytokine and chemokine production by primary keratinocytes lacking BRAF and RAF1.(A) Reduced ERK phosphorylation and increased JNK/p38 activation in primary delta/ deltaep2 keratinocytes stimulated with EGF and/or TNF alpha and IL1 beta for 15 min. (B) Increased cytokine and chemokine production in primary delta/ deltaep2 keratinocytes treated with EGF, TNF alpha and IL1 beta for 24 hr. Cytokine and chemokine production was determined by multiplex analysis, except for TSLP which was quantified by ELISA. Data represent mean ± SEM of 3–5 biological replicates. (C–D) Cells were pretreated with D-JNKI1 inhibitors prior to stimulation with EGF, TNF alpha and IL1 beta for 15 min (C) or 24 hr (D). Data represent the mean ± SEM of technical replicates (n = 3). (E–F) Effect of shRNA-mediated Mlk3 silencing on ERK and JNK phosphorylation and ICAM1 expression (E; stimulation with EGF, TNF alpha and IL1 beta for 15 min) and on the expression of Ccl2 and Tslp mRNA (F; stimulation with EGF, TNF alpha and IL1 beta for 24 hr) by F/F2 and delta/ deltaep2 keratinocytes. shRen, shRNA targeting Renilla, used as a control; sh1 and sh2, targeting Mlk3, binding sites nucleotide 2266–2285 and 2383–2402, respectively. The shRNAs were encoded by lentiviral vectors coexpressing GFP. GFP immunoblots are shown to confirm similar levels of infection in all samples. Data represent mean ± SEM of 4 biological replicates. Each keratinocyte culture represents a pool of three mice. Immunoblots are representative of three independent experiments. p1 = 0.041, p2 = 0.040, p3 = 1.89E-4, p4 = 0.018, p5 = 0.046, p6 = 0.020, p7 = 0.008, p8 = 0.016, p9 = 0.001, p10 = 0.018, p11 = 3.23E-4, p12 = 1.47E-4, p13 = 0.007, p14 = 0.03, p15 = 0.035, p16 = 0.023 and p17 = 0.046.DOI:https://dx.doi.org/10.7554/eLife.14012.018Compound knockdown (KD2) of BRAF and RAF1 induce the expression of inflammation markers by HaCat cells in a MLK3/JNK-dependent manner.(A) Reduced ERK and increased JNK/p38 activation in BRAF and RAF1 knockdown (KD2) HaCat cells stimulated with EGF, TNF alpha and IL1 beta for 15 min. (B) D-JNKI1 reduces ICAM1 and CCL2 (n = 4) expression in KD2 cells treated with TNF alpha. (C) MEKi induces ICAM1 and CCL2 (n = 3) expression in RAF1KD cells treated with TNF alpha. In (B–C), ICAM1 expression was measured after a 3 hr, CCL2 expression after a 24 hr treatment with TNF alpha. (D) Effect of MLK3 silencing on ERK and JNK phosphorylation in WT and KD2 cells stimulated as in (A). MLK3 was silenced using a pool of oligonucleotides targeting the following regions: 686–704; 1489–1507; 2122–2138; and 2348–2366. MLK3 KD cells stimulated as in (B–C) show a decrease in JNK activation, ICAM1 and CCL2 (n = 7) expression. Immunoblots are representative of three independent experiments. qPCR data represent mean ± SEM of three independent experiments run in duplicates (p1 = 4.62E-4, p2 = 0.013, p3 = 0.050, p4 = 8.60E-8, p5 = 0.050, p6 = 0.001, p7 = 0.001 and p8 = 0.012).DOI:https://dx.doi.org/10.7554/eLife.14012.019 Image collected and cropped by CiteAb from the following publication (https://elifesciences.org/articles/14012), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse ICAM-1/CD54 by Western Blot

Increased stress kinase signaling and JNK pathway-dependent cytokine and chemokine production by primary keratinocytes lacking BRAF and RAF1.(A) Reduced ERK phosphorylation and increased JNK/p38 activation in primary delta/ deltaep2 keratinocytes stimulated with EGF and/or TNF alpha and IL1 beta for 15 min. (B) Increased cytokine and chemokine production in primary delta/ deltaep2 keratinocytes treated with EGF, TNF alpha and IL1 beta for 24 hr. Cytokine and chemokine production was determined by multiplex analysis, except for TSLP which was quantified by ELISA. Data represent mean ± SEM of 3–5 biological replicates. (C–D) Cells were pretreated with D-JNKI1 inhibitors prior to stimulation with EGF, TNF alpha and IL1 beta for 15 min (C) or 24 hr (D). Data represent the mean ± SEM of technical replicates (n = 3). (E–F) Effect of shRNA-mediated Mlk3 silencing on ERK and JNK phosphorylation and ICAM1 expression (E; stimulation with EGF, TNF alpha and IL1 beta for 15 min) and on the expression of Ccl2 and Tslp mRNA (F; stimulation with EGF, TNF alpha and IL1 beta for 24 hr) by F/F2 and delta/ deltaep2 keratinocytes. shRen, shRNA targeting Renilla, used as a control; sh1 and sh2, targeting Mlk3, binding sites nucleotide 2266–2285 and 2383–2402, respectively. The shRNAs were encoded by lentiviral vectors coexpressing GFP. GFP immunoblots are shown to confirm similar levels of infection in all samples. Data represent mean ± SEM of 4 biological replicates. Each keratinocyte culture represents a pool of three mice. Immunoblots are representative of three independent experiments. p1 = 0.041, p2 = 0.040, p3 = 1.89E-4, p4 = 0.018, p5 = 0.046, p6 = 0.020, p7 = 0.008, p8 = 0.016, p9 = 0.001, p10 = 0.018, p11 = 3.23E-4, p12 = 1.47E-4, p13 = 0.007, p14 = 0.03, p15 = 0.035, p16 = 0.023 and p17 = 0.046.DOI:https://dx.doi.org/10.7554/eLife.14012.018Compound knockdown (KD2) of BRAF and RAF1 induce the expression of inflammation markers by HaCat cells in a MLK3/JNK-dependent manner.(A) Reduced ERK and increased JNK/p38 activation in BRAF and RAF1 knockdown (KD2) HaCat cells stimulated with EGF, TNF alpha and IL1 beta for 15 min. (B) D-JNKI1 reduces ICAM1 and CCL2 (n = 4) expression in KD2 cells treated with TNF alpha. (C) MEKi induces ICAM1 and CCL2 (n = 3) expression in RAF1KD cells treated with TNF alpha. In (B–C), ICAM1 expression was measured after a 3 hr, CCL2 expression after a 24 hr treatment with TNF alpha. (D) Effect of MLK3 silencing on ERK and JNK phosphorylation in WT and KD2 cells stimulated as in (A). MLK3 was silenced using a pool of oligonucleotides targeting the following regions: 686–704; 1489–1507; 2122–2138; and 2348–2366. MLK3 KD cells stimulated as in (B–C) show a decrease in JNK activation, ICAM1 and CCL2 (n = 7) expression. Immunoblots are representative of three independent experiments. qPCR data represent mean ± SEM of three independent experiments run in duplicates (p1 = 4.62E-4, p2 = 0.013, p3 = 0.050, p4 = 8.60E-8, p5 = 0.050, p6 = 0.001, p7 = 0.001 and p8 = 0.012).DOI:https://dx.doi.org/10.7554/eLife.14012.019 Image collected and cropped by CiteAb from the following publication (https://elifesciences.org/articles/14012), licensed under a CC-BY license. Not internally tested by R&D Systems.