antitrpc5 rabbit polyclonal  (Alomone Labs)

Journal: bioRxiv

Article Title: Cav3.1 is a leucine sensor in POMC neurons mediating appetite suppression and weight loss

doi: 10.1101/2024.09.13.612843

Figure Lengend Snippet: Molecular profiling of hypothalamic leucine-sensing neurons identifies Cacnag1 as a top marker ( A ) PhosphoTRAP strategy for identification of leucine-responsive neurons in the MBH. ( B ) Volcano plot of differentially enriched markers of MBH leucine-responsive neurons. Cacna1g and Trpc5 are highlighted. ( C-D ) RNAscope fluorescent in situ hybridisation (FISH) of Cacna1g , cfos and Pomc mRNAs in MBH after MBH leucine administration. ( C ) Representative images: white squares: regions shown in the zoom-in images; white arrows: Cacna1g +/POMC neurons expressing cfos; cale bar: 200 μm; 3V: third ventricle. ( D ) Quantification of imaging analysis. n = 4 for aCSF; n = 6 for Leu. ****p<0.0001. Values are reported as mean ± SEM. ( E-F ) RNAscope FISH of Cacna1g , Trpc5 and Pomc mRNAs in MBH. ( E ) Representative images: white square: region shown in the zoom-in images; white arrows: Cacna1g +/ Trpc5 + double positive POMC neurons; scale bar: 200um; 3V: third ventricle. ( F ) Relative proportion of MBH POMC neurons expressing Cacna1g and Trpc5 . n = 1647 POMC neurons from 5 WT mice.

Article Snippet: The following primary antibodies were used: anti-Cav3.1 (C-terminal) mouse monoclonal (1:1000; abcam, Cat# ab134269), anti-TRPC5 rabbit polyclonal (1:1000; Alomone Labs, Cat# ACC-020), anti-GAPDH mouse monoclonal (1:1000; Santa Cruz Biotechnology, Cat# sc-32233), anti-phospho-p70 S6 Kinase (Thr389) rabbit polyclonal (1:1000; Cell Signalling Technology, Cat# 9205), anti-P70 S6K rabbit monoclonal (1:1000; Cell Signalling Technology, Cat# 2708) and anti-HA mouse monoclonal (1:1000; Cell Signalling Technology, Cat# 2367).

Techniques: Marker, RNAscope, In Situ, Hybridization, Expressing, Imaging

Journal: bioRxiv

Article Title: Cav3.1 is a leucine sensor in POMC neurons mediating appetite suppression and weight loss

doi: 10.1101/2024.09.13.612843

Figure Lengend Snippet: Additional data related to . (A) Representative immunofluorescence confocal image showing extensive co-localisation of phosphorylated rpS6 and cFos in the ARH and VMH after MBH leucine injection. Scale bar: 50 μm. (B) Abundance of hypothalamic marker transcripts in MBH input samples from both aCSF and leucine groups. (C) Enrichment (I.P./Input) of activity dependent genes in MBH samples from both aCSF and leucine groups compared to Gapdh . (D) Validation of PhosphoTRAP RNAseq results with RT-qPCR of 41 selected genes. A statistically significant positive linear relationship is found between these two RNA quantification techniques. n = 4 for RNAseq, n = 3-4 for RT-qPCR. (E) Ingenuity Pathway analysis (IPA) of differentially enriched canonical pathways in leucine-responsive neurons identified in PhosphoTRAP assay. See Table S5 for full data. (F) Normalised fold enrichment ratio of Cacna1g analysed by RT-qPCR. The RNA samples used were from the same experiment also submitted for RNAseq. n = 3 for aCSF; n = 4 for Leu. (G-I) Quantification RNAscope analysis after MBH leucine injection as shown in . ( G ) % all MBH cells expressing cfos , ( H ) % all POMC neurons expressing cfos , ( I ) number of Cacna1g spots per POMC neuron. n = 4 for aCSF; n = 6 for Leu. (J) Relative proportion of all MBH cells expressing Cacna1g and Trpc5 . N = 59060 MBH cells from 5 WT mice. (K) Representative Western blot of co-immunoprecipitation assay of heterologously expressed human TRPC5 and Cav3.1 in HEK293 cells. This experiment has been repeated twice with similar results. *p<0.05, ***p<0.001, ****p<0.0001. Values are reported as mean ± SEM

Article Snippet: The following primary antibodies were used: anti-Cav3.1 (C-terminal) mouse monoclonal (1:1000; abcam, Cat# ab134269), anti-TRPC5 rabbit polyclonal (1:1000; Alomone Labs, Cat# ACC-020), anti-GAPDH mouse monoclonal (1:1000; Santa Cruz Biotechnology, Cat# sc-32233), anti-phospho-p70 S6 Kinase (Thr389) rabbit polyclonal (1:1000; Cell Signalling Technology, Cat# 9205), anti-P70 S6K rabbit monoclonal (1:1000; Cell Signalling Technology, Cat# 2708) and anti-HA mouse monoclonal (1:1000; Cell Signalling Technology, Cat# 2367).

Techniques: Immunofluorescence, Injection, Marker, Activity Assay, Quantitative RT-PCR, RNAscope, Expressing, Western Blot, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Cav3.1 is a leucine sensor in POMC neurons mediating appetite suppression and weight loss

doi: 10.1101/2024.09.13.612843

Figure Lengend Snippet: (A-D) Calcium imaging assay on mouse acute primary cultured hypothalamic POMC neurons. Representative calcium trace of a POMC neuron activated by leucine co-treated with vehicle ( A ) and Cav3.1 inhibitor TTA-P2 ( B ). Quantification of activated POMC neurons ( C ) and all cells ( D ) recorded over 4 independent experiments. (E-H) Calcium imaging assay on hIPSC derived POMC neurons. Representative calcium trace of a hIPSC derived neuron activated by leucine co-treated with vehicle ( E ) and TTA-P2 ( F ). Quantification of hIPSC derived POMC neuron ( G ) and all cells ( H ) recorded over 4 independent experiments. (I-K) Brain slice current-clamp recordings of arcuate POMC neurons with leucine treatment. ( I ) Representative current-clamp trace depicting a characteristic depolarization of arcuate POMC neurons by 1 mM leucine. ( J ) Histogram summarising the acute effect of leucine on the membrane potential of POMC neurons (n=19). ( K ) Box plot of action potential frequency (ARF) in a subpopulation of POMC neurons over time (n=6). (L-N) Brain slice current-clamp recordings of arcuate POMC neurons with TTA-P2 pre-treatment prior leucine. ( L ) Representative trace showing that 1 mM leucine fails to induce a depolarization of POMC neurons exposed to 10 μM TTA-P2. ( M ) Histogram summarizing the acute effect of leucine on the membrane potential of POMC neurons in the presence of 10 μM TTA-P2 (n=5). ( N ) Box plot of APF of POMC neurons in the presence of TTA-P2 over time (n=4). (O-Q) Brain slice current-clamp recordings of arcuate POMC neurons from Trpc5 KO mice with leucine treatment. ( O ) Representative trace showing that 1 mM leucine fails to induce a depolarization of POMC neurons on a Trpc5 KO background. ( P ) Histogram summarizing the acute effect of leucine on the membrane potential of POMC neurons deficient for TRPC5 (n=14). ( Q ) Box plot of APF of POMC neurons deficient for TRPC5 over time (n=13). (R-S) Feeding responses assessment after MBH leucine injection on awake mice. ( R ) Diagram of the experimental paradigm. ( S ) Acute feeding response after MBH leucine co-treated with vehicle or TTA-P2. n = 10 per group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Range bars in box plots (K, N, Q) indicates min to max and comparisons were made versus the minute bin prior leucine treatment. Values in (S) are reported as mean ± SEM.

Article Snippet: The following primary antibodies were used: anti-Cav3.1 (C-terminal) mouse monoclonal (1:1000; abcam, Cat# ab134269), anti-TRPC5 rabbit polyclonal (1:1000; Alomone Labs, Cat# ACC-020), anti-GAPDH mouse monoclonal (1:1000; Santa Cruz Biotechnology, Cat# sc-32233), anti-phospho-p70 S6 Kinase (Thr389) rabbit polyclonal (1:1000; Cell Signalling Technology, Cat# 9205), anti-P70 S6K rabbit monoclonal (1:1000; Cell Signalling Technology, Cat# 2708) and anti-HA mouse monoclonal (1:1000; Cell Signalling Technology, Cat# 2367).

Techniques: Imaging, Cell Culture, Derivative Assay, Slice Preparation, Membrane, Injection