Journal: Biomedicines

Article Title: Klotho Modulates Pro-Fibrotic Activities in Human Atrial Fibroblasts through Inhibition of Phospholipase C Signaling and Suppression of Store-Operated Calcium Entry

doi: 10.3390/biomedicines10071574

Figure Lengend Snippet: Effects of Klotho (100 pM) and selective TRPC6 inhibitor (BI-749327, 1 μM) on ionic currents of transient receptor potential canonical channel (I TRPC ) in human atrial fibroblasts. ( A ) The current tracings and I–V relationship of I TRP showed that BI-749327-treated fibroblasts (1 μM, 48 h, n = 13–15 from 4 independent experiments), Klotho-treated fibroblasts (100 pM, 48 h, n = 15 from 3 independent experiments), and combined Klotho and BI-749327 ( n = 15 from 3 independent experiments) had similarly lower peak inward currents and similarly lower peak outward currents compared with the control ( n = 13 from 4 independent experiments). ( B ) Western blots showed a similar expression of TRPC6 and total phospholipase C (PLC) between fibroblasts treated with Klotho (100 pM, 48 h) and control fibroblasts. However, the phosphorylated PLC (p-PLC) expression was lower in the fibroblasts treated with Klotho (100 pM, 48 h) ( n = 3 from 3 independent experiments). ( C , D ) Example photographs and average data ( n = 9 from 3 independent experiments) showed that human atrial fibroblasts treated with BI-749327 (1 μM, 48 h), Klotho (100 pM, 48 h), and combined Klotho and BI-749327 exhibited similarly less migratory capability than the control. Arrows indicate migrating fibroblasts in the wound space. GAPDH was used as a loading control. PLCβ3, phospholipase C beta 3; pPLCβ3, phosphorylated phospholipase C beta 3; TRPC6, transient receptor potential canonical channel 6. * p < 0.05, *** p < 0.005.

Article Snippet: We probed all blots with primary antibodies against procollagen type IA1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), procollagen type III (Abcam, Cambridge, UK), α-smooth muscle actin (α-SMA) (Abcam, Cambridge, UK), TRPC6 (Alomone Labs, Jerusalem, Israel), human PLC beta 3 (PLCβ3) (R&D Systems, Minneapolis, MN, USA), phospho-PLC beta 3 (Ser537) (pPLCβ3 Ser 537) (Cell Signaling Technology, Beverly, MA, USA); secondary antibodies were all conjugated with horseradish peroxidase.

Techniques: Control, Western Blot, Expressing

Journal: The Journal of Physiology

Article Title: Purinergic receptor stimulation induces calcium oscillations and smooth muscle contraction in small pulmonary veins

doi: 10.1113/JP274731

Figure Lengend Snippet: A, representative experiment showing ATP‐induced SPV contraction in the absence of PLC inhibitor and in the presence of PLC inactive (U73343 10 μm) and active (U73122 10 μm) inhibitor analogues. B, summary of the effect of the PLC inactive and active inhibitor analogues on ATP‐induced SPV contraction. The data are expressed as the mean ± SEM (n = 6 from three rats) of the percentage of maximal ATP‐induced SPV contraction in the absence of PLC inhibitor. ***P < 0.001; NS, not significant (one‐way ANOVA followed by Tukey's comparison). C, representative experiment showing SPV contraction in response to ATP (50 μm) in sHBSS and in Ca2+‐free sHBSS (0‐Ca2+). SPV contraction was sustained or transient in the presence or absence of extracellular Ca2+, respectively. The trace is presentative of eight experiments from different slices from five rats.

Article Snippet: These drugs were used at 10 μ m because, at this concentration, {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}} U73122 produces a submaximal inhibition of vasoconstriction in systemic arteries by inhibiting PLC‐β (Kuang et al . 2017 ).

Techniques: Analogues, Comparison

Journal: Frontiers in Neuroanatomy

Article Title: Up-regulation of CB 1 cannabinoid receptors located at glutamatergic terminals in the medial prefrontal cortex of the obese Zucker rat

doi: 10.3389/fnana.2022.1004702

Figure Lengend Snippet: Primary antibodies used.

Article Snippet: CB 1 cannabinoid receptor, G i/o α-subunits, and phospholipase C-β 1 (PLC-β 1 ) were detected by immunoblotting with the following antibodies: goat polyclonal anti-CB 1 receptor (CB1-Go-Af450; Frontier Science Co. Ltd, Hokkaido, Japan), rabbit polyclonal anti-Gα o (sc-387; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-Gα i 1 (sc-391; Santa Cruz Biotechnology), rabbit polyclonal anti-Gα i 2 (sc-7276; Santa Cruz Biotechnology), rabbit polyclonal anti-Gα i 3 (sc-262; Santa Cruz Biotechnology) and mouse monoclonal anti-PLC-β 1 (BD Transduction Laboratories, San Diego, CA, USA) ( ).

Techniques: Affinity Purification, Transduction