Journal: Molecular Pain

Article Title: Cannabinoid-mediated modulation of neuropathic pain and microglial accumulation in a model of murine type I diabetic peripheral neuropathic pain

doi: 10.1186/1744-8069-6-16

Figure Lengend Snippet: During 8 months of diabetes, Western blotting (A) identified mild increasing levels of Iba1 (B), beginning at 3 months of diabetes, when compared to non-diabetic littermates . As well, phosphorylated p38 MAPK elevates during diabetes until 5 months of time ( C ). Sample protein blots are demonstrated in A from a total of 3 sample blots identified for each marker and each time point. Multiple ANOVA tests were performed in each case, with * indicating significant difference (p < 0.05) between diabetic and non-diabetic cohorts.

Article Snippet: Primary antibodies used were goat anti-ionized calcium-binding adaptor molecule 1 (Iba-1; 1:1000; Abcam, Cambridge, MA) for microglial identification, rabbit anti-cannabinoid receptor 1 antibody (CB1 receptor, 1:500, Sigma Aldrich Canada), rabbit anti-cannabinoid receptor 2 antibody (CB2 receptor, 1:500, Sigma Aldrich Canada), rabbit anti-phosphorylated p38 Mitogen Activated Protein Kinase (p-p38 MAPK; 1:100; Cell Signalling Technology, USA), and rabbit anti-microtubule associated protein-2 antibody produced in rabbit (MAP-2, 1:500, M3696, Sigma Aldrich Canada) for neuronal identification.

Techniques: Western Blot, Marker

Journal: Molecular Pain

Article Title: Cannabinoid-mediated modulation of neuropathic pain and microglial accumulation in a model of murine type I diabetic peripheral neuropathic pain

doi: 10.1186/1744-8069-6-16

Figure Lengend Snippet: During 5 months of diabetes, Western blotting (A) identified mild increased levels of Iba1 (B) in diabetic mice . As well, phosphorylated p38 MAPK was elevated during diabetes at 5 months of time ( C ). Sample protein blots are demonstrated in A (total of 3 sample blots identified for each marker and each intervention at the final time point). Only intranasal or intraperitoneal cannabidiol administered at the onset of diabetes was associated with suppression of Iba1 and phosphorylated p38 MAPK levels at endpoint ( B, C ). Multiple ANOVA tests were performed in each case, with * indicating significant difference (p < 0.05) between diabetic cohorts receiving intranasal cannabidiol and no intervention after 5 months of diabetes.

Article Snippet: Primary antibodies used were goat anti-ionized calcium-binding adaptor molecule 1 (Iba-1; 1:1000; Abcam, Cambridge, MA) for microglial identification, rabbit anti-cannabinoid receptor 1 antibody (CB1 receptor, 1:500, Sigma Aldrich Canada), rabbit anti-cannabinoid receptor 2 antibody (CB2 receptor, 1:500, Sigma Aldrich Canada), rabbit anti-phosphorylated p38 Mitogen Activated Protein Kinase (p-p38 MAPK; 1:100; Cell Signalling Technology, USA), and rabbit anti-microtubule associated protein-2 antibody produced in rabbit (MAP-2, 1:500, M3696, Sigma Aldrich Canada) for neuronal identification.

Techniques: Western Blot, Marker

Journal: Frontiers in Immunology

Article Title: Endoplasmic reticulum stress promoted acinar cell necroptosis in acute pancreatitis through cathepsinB-mediated AP-1 activation

doi: 10.3389/fimmu.2022.968639

Figure Lengend Snippet: ER stress inhibition alleviated CTSB maturation and PKCα-JNK-cJun-mediated AP-1 activation in AP. (A-C) In vitro , Pancreatic acinar cells were pre-treated with ER stress inhibitor 4-PBA (2.5 μM, 5 μM, 10 μM) for 30 min and then stimulated by 200 nM CCK for 6 h. (A) Immunoblot analysis of CTSB levels in pancreatic acinar cells. (B) Immunoblot analysis of p-PKCα, p-JNK, p-ERK, p-p38-MAPK and p-cJun levels in pancreatic acinar cells. (C) EMSA analysis of AP-1 binding ability in pancreatic acinar cells. (D) In vivo , AP was induced by injection of caerulein (100 μg·kg -1 ) and LPS (5 mg·kg -1 ) in Balb/C mice, and treated with 4-PBA (4 mg per mouse, i.p., 0.5 h before the first caerulein injection). Immunoblot analysis of p-PKCα, p-JNK, p-ERK, p-p38-MAPK and p-cJun levels of pancreatic tissue in mice. (E) In vivo , AP was induced by injection of L-arginine (10 mg·kg -1 ) in Balb/C mice, and treated with 4-PBA (4 mg per mouse, i.p., 0.5 h before the first L-arginine injection). Immunoblot analysis of p-PKCα, p-JNK, p-ERK, p-p38-MAPK and p-cJun levels of pancreatic tissue in mice. All experiments were performed at least three times. Data are presented as Mean ± SEM. n = 6 per group. * p < 0.05 versus NC, # p < 0.05 versus CCK or AP. Cae, caerulein; CCK, cholecystokinin; CTSB, cathepsin B; LPS, lipopolysaccharid; L-Arg, L-arginine; NC, normal control.

Article Snippet: Antibodies against C/EBP homology protein (CHOP, cat # 2895), cathepsin B (CTSB, cat # 31718), phosphorylated protein kinase α (p-PKCα, cat # 9375), phosphorylated c-Jun NH2-terminal kinase (p-JNK, cat # 4668), phosphorylated extracellular signal-regulated kinas (p-ERK, cat # 4370), phosphorylated p38 mitogen activated protein kinases (p-p38MAPK, cat # 4511), phosphorylated cJun (p-cJun, cat # 3270) and IL1β(3A6) (cat # 12242) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Activation Assay, In Vitro, Western Blot, Binding Assay, In Vivo, Injection

Journal: Journal of Cellular and Molecular Medicine

Article Title: The mechanisms and significance of up‐regulation of RhoB expression by hypoxia and glucocorticoid in rat lung and A549 cells

doi: 10.1111/jcmm.12809

Figure Lengend Snippet: Activation of HIF ‐1α, ERK and JNK , but not p38 MAPK are involved in hypoxia‐induced RhoB expression in A549 cells. ( A and B ) A549 cells (1.0 × 10 6 per well) were cultured for overnight and were pre‐incubated with or without HIF ‐1α inhibitor 400083 (100 μM) for 1 hr prior to hypoxic exposure for 12 hrs ( A ) or 8 hrs ( B ). HIF ‐1α and RhoB proteins were assessed by Western blot and β‐actin was used as a loading control. ( B ) RhoB mRNA was assessed by qRT ‐ PCR and β‐actin was used as a normalization control. ( C ) A549 cells were exposed to hypoxia for indicated times, then p‐ JNK , JNK , p‐ ERK , ERK , p‐p38 and p38 were detected by Western blot. ( D ) A549 cells were pre‐incubated with or without SB 203580 (10 μM), SP 600125 (20 μM) or PD 098059 (20 μM) for 1 hr prior to hypoxic exposure for 12 hrs, and RhoB protein was assessed by Western blot. The protein band was quantified by densitometric analysis. The value represents mean (±S.D.) of increased folds versus normoxic vehicle control from three independent experiments. Symbols & and # represent P < 0.05 and P < 0.01 compared with control cells respectively.

Article Snippet: Briefly, protein extracts were separated by SDS‐PAGE, transferred to nitrocellulose membrane (Millipore, Ireland) and probed overnight with primary antibodies against RhoB (sc‐180; Santa Cruz Biotechnology, Santa Cruz, TX, USA), β‐actin (A5441; Sigma‐Aldrich Chemicals), HIF‐1α (H‐206; Santa Cruz Biotechnology), phosphorylated JNK, JNK, phosphorylated ERK, ERK, phosphorylated p38 mitogen‐activated protein kinase (MAPK) or p38 MAPK (Cell Signaling, Danvers, MA, USA).

Techniques: Activation Assay, Expressing, Cell Culture, Incubation, Western Blot, Quantitative RT-PCR