Journal: Cellular signalling

Article Title: Cannabinoid receptor 1 suppresses transient receptor potential vanilloid 1-induced inflammatory responses to corneal injury

doi: 10.1016/j.cellsig.2012.10.015

Figure Lengend Snippet: CB1 and TRPV1 protein–protein interaction. (A) Anti-TRPV1 antibody selectivity validation. Western blot detects TRPV1 protein expression with apparent molecular weight in agreement with previous reports. It was selectively suppressed by 74% decline following TRPV1 siRNA transfection of HCEC. (B) CB1-induces decline in TRPV1 phosphorylation status. Western blot analysis of TRPV1 immunopreciptiates using an anti-phospho-threonine antibody revealed that 30 min 10 μM WIN preincubation dampened TRPV1 phosphorylation. Anti-phosphothreonine antibody specificity was validated based on blot with the positive control provided by the commercial source. (C) Anti-CB1 antibody selectivity validation. Western blot detects CB1 protein expression with apparent molecular weight in agreement with previous reports. It was selectively suppressed by 76% decline following CB1 siRNA transfection. (D) TRPV1 complexation with CB1 and TAK1. Co-immunoprecipitation of whole cell lysates obtained with either an anti-TAK1, CB1 or TRPV1 antibody following CAP (10 μM) exposure. Immunocomplexes were probed for TRPV1 content with Western blot analysis using an anti-TRPV1 antibody. (E) CB1 complexation with TRPV1 and TAK1. Co-immunoprecipitation of whole cell lysates obtained with either an anti-TAK1, CB1 or TRPV1 antibody following CAP (10 μM) exposure. Immunocomplexes were probed for CB1 content with Western blot analysis using an anti-CB1 antibody. Antibody selectivity was validated by showing that the IgG immunoprecipitated complexes yielded no immunoreactive bands. Results are expressed as means±SEM (n=3). Differences of p<0.05 were considered as significant.

Article Snippet: The immune reactivity was detected with a Western blot analysis kit (Amersham ECL Plus; GE Healthcare Life Sciences, Piscataway, NJ).

Techniques: Biomarker Discovery, Western Blot, Expressing, Molecular Weight, Transfection, Phospho-proteomics, Positive Control, Immunoprecipitation

Journal: Cellular signalling

Article Title: Cannabinoid receptor 1 suppresses transient receptor potential vanilloid 1-induced inflammatory responses to corneal injury

doi: 10.1016/j.cellsig.2012.10.015

Figure Lengend Snippet: Blunting by CB1 activation of TRPV1-induced JNK1 and TAK1 phosphorylation. (A) TRPV1 and CB1 mediated JNK1/2 phosphorylation. Western blot analysis with anti-phospho JNK1/2 antibody reveals increases in JNK1/2 phosphorylation induced by 5 min exposure to CAP (20 μM). Such rises were inhibited by 30 min preexposure to either WIN (10 μM), or CPZ (10 μM). Pre-incubation with AM251 (10 μM), for 30 min prior to WIN (10 μM), exposure suppressed declines in CAP (20 μM)-induced JNK1/2 phosphorylation induced by WIN (10 μM). (B) Dependence of WIN blunting of TRPV1-induced TAK1 phosphorylation on CB1 protein expression. CAP (10 μM)-induced increases in TAK1 phosphorylation at 5 min in cells transduced with scrambled siRNA (left panel) are compared with those in CB1 siRNA transduced cells (right panel). WIN-induced suppression of TRPV1-induced TAK1 phosphorylation was eliminated in CB1 siRNA transduced cells (last lane on the right). The results are shown as means±SEM (n=3, p<0.05).

Article Snippet: The immune reactivity was detected with a Western blot analysis kit (Amersham ECL Plus; GE Healthcare Life Sciences, Piscataway, NJ).

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Incubation, Expressing, Transduction