cav3 1  (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: 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: In brief, 33.3 μl beads per sample were washed with 1 mL TBS with 1% Triton X-100 (TBST) then blocked with 500 μl TBST with 1μg/ml BSA for 20 min at 4°C with rotation, followed by incubating with 1 μg N-terminal anti-Cav3.1 (Alomone Labs, Cat# ACC-021) (or 1 μg rabbit IgG as a control) in 500 μl lysis buffer with 1 μg/ml BSA at 4°C with rotation for 1 hr.

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: In brief, 33.3 μl beads per sample were washed with 1 mL TBS with 1% Triton X-100 (TBST) then blocked with 500 μl TBST with 1μg/ml BSA for 20 min at 4°C with rotation, followed by incubating with 1 μg N-terminal anti-Cav3.1 (Alomone Labs, Cat# ACC-021) (or 1 μg rabbit IgG as a control) in 500 μl lysis buffer with 1 μg/ml BSA at 4°C with rotation for 1 hr.

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

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-F) Fluo8 calcium flux assay of HEK293-hCav3.1 cells. ( A-B ) Calcium responses and AUC quantification after KCl and Leu treatments. ( C-D ) Calcium responses and AUC quantification after Leu, Val and Ile treatments. ( E-F ) Calcium responses and AUC quantification after Leu treatment with or without mTOR inhibitor Torin 1 (100 nM) pre-treatment. n = 6 per group merged from 2 independent experiments with triplicates each. ( G-J) Whole-cell patch-clamp recording of HEK293 transiently expressing hCav3.1 treated with leucine. ( G ) Representative traces of Ca 2+ currents recorded in vehicle solution (left) and after application of 1 mM leucine. In both cases, cells were held at −100 mv and increasing steps of 10 mV were applied from −90 to +20 mv. ( H ) Normalised current-voltage (I-V) curves of HEK293-hCav3.1 cells treated with vehicle or leucine under the activation protocol. Stead-state activation (G/G max ) and inactivation (I/I max ) curves ( I ) and V 50 histogram ( J ) of HEK293-hCav3.1 cells treated with vehicle or leucine. Veh_act: n=13, Leu_act: n=8, Veh_inact: n=13, Leu_inact: n=6. (K-L) In vitro binding assay of hCav3.1 to fluorescent labelled leucine ( K ) and valine ( L ). Post-assay Western blots below the plots validate successful immunoprecipitation (IP). IgG: unimmunized rabbit IgG control; DN: heat-denatured cell lysate prior IP; WT: cell lysate from non-transfected HEK293 cells. Data represent 3 technical replicates upon fluorescence measurements. These experiments has been repeated twice with similar results. Data of representative sets of experiment are shown. (M) Human Cav3.1 protein (PDB id: 6KZO) is shown in cartoon representation with domain I, II, III and IV coloured in red, light blue, yellow and green, respectively. The representative binding poses of L-leucine are shown in sphere representation with poses relevant to different sites coloured distinctly: yellow (putative site I, marked with an orange circle), green (putative site II, marked with a green circle), red (putative site III, marked with a red circle) and light pink (putative site IV, marked with a blue circle). The figure was generated in PyMol 2.5 (Schrodinger LLC). (N) Schematic representation of hCav3.1 critical amino acid residues of corresponding predicted leucine binding sites chosen for mutagenesis experiments, colour-coded as in ( M ). (O) 2D ligand interactions diagrams for the docked poses of leucine at Site II. The residues mutated for experimental validation are shown with asterisks. The dotted arrow signs indicate hydrogen bonding whilst the dotted contour around the ligand pose indicate hydrophobic interactions with residues shown. These diagrams were generated in MOE. (P-Q) Fluo8 calcium flux assay of HEK293-hCav3.1 Site II mutant (V841A/V845A/L851A) cells. Calcium responses ( P ) and AUC quantification ( Q ) after KCl and Leu treatments. n=8 merged from 4 independent experiments with duplicates each. (R) In vitro fluorescent leucine binding assay of WT and Site I–IV hCav3.1 mutants. Post-assay Western blot below the plot validates successful IP. Data represent 3 technical replicates upon fluorescence measurements. These experiments have been repeated twice with similar results. Data of a representative experiment is shown. Groups denoted with different letters in (B, D, F) indicate significant difference (p<0.05). *p<0.05, **p<0.01, ****p<0.0001. Values and calcium response traces in (A, C, E) are reported as mean ± SEM.

Article Snippet: In brief, 33.3 μl beads per sample were washed with 1 mL TBS with 1% Triton X-100 (TBST) then blocked with 500 μl TBST with 1μg/ml BSA for 20 min at 4°C with rotation, followed by incubating with 1 μg N-terminal anti-Cav3.1 (Alomone Labs, Cat# ACC-021) (or 1 μg rabbit IgG as a control) in 500 μl lysis buffer with 1 μg/ml BSA at 4°C with rotation for 1 hr.

Techniques: Calcium Flux Assay, Patch Clamp, Expressing, Activation Assay, In Vitro, Binding Assay, Western Blot, Immunoprecipitation, Control, Transfection, Fluorescence, Generated, Mutagenesis

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-F) Fluo8 calcium flux assay of HEK293 (-hCa v 3.1 and -WT) cells. Dose-dependent KCl-induced calcium responses and AUC quantification of HEK293-hCa v 3.1 ( A-B ); Lack of calcium responses in non-transfected HEK293-WT cells after various doses of KCl ( C-D ) and leucine treatment ( E-F ). n = 3 per group. (G-I) In vitro fluorescent leucine binding assay of individual hCa v 3.1 domains. Schematic diagram of recombinant truncated hCav3.1 domain fragments construction ( G ). Quantification of fluorescence measurements for fluorescent leucine binding ( H ). Post-assay Western blot validates successful expression and immunoprecipitation of the recombinant domain fragments ( I ). Red asterisks indicate expected band size of the fragments. Note that this assay was performed on transiently transfected cells; the full-length hCa v 3.1 expression was far weaker yet bound stronger to FAM-leucine compared to the truncated fragments. Data represent 3 technical replicates upon fluorescence measurements. These experiments have been repeated twice with similar results. Data of a representative experiment is shown. (J-K) Cav3.1 contributes to leucine induced mTOR1 activation in HEK293-hCav3.1 cells. ( J ) Representative Western blot of Thr389 phosphorylation of S6K in HEK293-hCav3.1 cells treated with leucine. with or without co-treatment of TTA-P2. ( K ) Normalised densitometric quantification of pThr389 S6K to total S6K ratio from the same blot. Data were merged from 2 independent experiments with duplicates each. (L) Western blot validation for the protein expression of hCav3.1 Site I/II/III/IV mutants in respective stably expressing HEK293 cells. (M, P, S) 2D ligand interactions diagrams for the docked poses of L-Leu at Site I ( M ), Site III ( P ) and Site IV ( S ). The residues mutated for experimental validation are shown with asterisks. The dotted arrow signs indicate hydrogen bonding whilst the dotted contour around the ligand pose indicate hydrophobic interactions with residues shown. (N-O, Q-R, T-U) Fluo8 calcium flux assay of HEK293-hCa v 3.1 predicted Leu-binding mutant cells. Calcium responses and AUC quantification after KCl and Leu treatments of Site I mutant cells (M948A/T949A; N-O ); Site III mutant cells (E789A/V815A/R846A; Q-R ) and Site IV mutant cells (D902A/K927A; T-U ). n = 8 merged from 4 independent experiments with duplicates each. (V) Amino acid sequence alignments of the domain II and III voltage-sensing S4 transmembrane segments of human Cav3.1 against hCav3.2 and hCav3.3 (upper panel) and against Cav3.1 of various mammalian species and zebrafish (lower panel). Bolded underlined black “R”: consensus voltage-sensing arginine residues; bolded red: critical residues predicted to mediate leucine binding and were chosen for mutagenesis in this study; orange: other residues predicted to participate in leucine binding; bolded blue: residues different from human Cav3.1. Groups denoted with different letter in (B,L) indicate significant difference (p<0.05). *p<0.05, **p<0.01, ***p<0.01, ****p<0.0001. Values and calcium response traces in (A, C, E, N, Q, T) are reported as mean ± SEM.

Article Snippet: In brief, 33.3 μl beads per sample were washed with 1 mL TBS with 1% Triton X-100 (TBST) then blocked with 500 μl TBST with 1μg/ml BSA for 20 min at 4°C with rotation, followed by incubating with 1 μg N-terminal anti-Cav3.1 (Alomone Labs, Cat# ACC-021) (or 1 μg rabbit IgG as a control) in 500 μl lysis buffer with 1 μg/ml BSA at 4°C with rotation for 1 hr.

Techniques: Calcium Flux Assay, Transfection, In Vitro, Binding Assay, Recombinant, Fluorescence, Western Blot, Expressing, Immunoprecipitation, Activation Assay, Stable Transfection, Mutagenesis, Sequencing

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) Diagram of experimental strategy to generate Cacna1g MBH KO mice. (B-C) Western blot analysis of the CRISPR knockout efficiency of Cav3.1 in Cacna1g MBH KO mediobasal hypothalamus with a C-terminal specific Cav3.1 antibody. GAPDH from the same blot was used as a loading control. Representative image ( B ) and densitometric quantification ( C ) of the blots. n = 18 per group. (D) Representative DNA electrophoresis image of T7 endonuclease I mutation detection assay of genomic DNA isolated from the MBH tissues of Con and Cacna1g MBH KO mice. Black arrow indicates parental unmodified PCR amplicons and red arrows indicate digested mutated fragments of expected size. (E) Acute feeding response after MBH leucine injection in Con and Cacna1g MBH KO . Con: n = 11; MBH KO: n=12. (F) Body weight change of Con and Cacna1g MBH KO over the course of 4 weeks feeding on 14% normal protein (P14) diet and 45% hight protein (P45) diet. (G-J) Feeding behaviour assessments of Con and Cacna1g MBH KO mice on P14 and P45 diets during indirect calorimetry measurement. ( G ) 24-hr profile of cumulative energy intake, ( H ) 24-hr total energy intake, ( I ) 24-hr average meal size and ( J ) 24-hr average meal number. (K-M) Energy expenditure of Con and Cacna1g MBH KO mice on P14 and P45 diets during indirect calorimetry measurement. ANCOVA analysis of average 24-hr energy expenditure against body weight of Con and Cacna1g MBH KO on P14 ( K ) and P45 ( L ) diets, 24-hr average energy expenditure ( M ). For ( F-M ) Con-P14: n = 6; Con-P45: n = 6; KO-P14: n = 7; KO-P45: n = 7. *p<0.05, **p,0.01, ***p<0.001. Values are reported as mean ± SEM.

Article Snippet: In brief, 33.3 μl beads per sample were washed with 1 mL TBS with 1% Triton X-100 (TBST) then blocked with 500 μl TBST with 1μg/ml BSA for 20 min at 4°C with rotation, followed by incubating with 1 μg N-terminal anti-Cav3.1 (Alomone Labs, Cat# ACC-021) (or 1 μg rabbit IgG as a control) in 500 μl lysis buffer with 1 μg/ml BSA at 4°C with rotation for 1 hr.

Techniques: Western Blot, CRISPR, Knock-Out, Control, Nucleic Acid Electrophoresis, Mutagenesis, Detection Assay, Isolation, Injection

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) Diagram of experimental strategy to generate Cacna1g POMC KO mice. (B-C) Confocal i mmunofluorescence (IF) microscopy analysis of the CRISPR knockout efficiency of Cav3.1 in Cacna1g POMC KO mediobasal hypothalamus with a C-terminal specific Cav3.1 antibody validated in Fig. S3F. ( B ) Zoom-in images of the regions highlighted with white squares in (Fig. S6A). White arrows with solid line: example POMC+/mCherry+/Cav3.1+ neurons. Blue arrows with broken line: example POMC+/mCherry+/Cav3.1-neurons. Scale bar: 50 µm. ( C ) Quantification for % mCherry+ transduced POMC neurons expressing Cav3.1. Con: n = 5; Cacna1g POMC KO : n = 6. (D) Acute feeding response after MBH leucine injection in Con and Cacna1g POMC KO . Con: n = 10; KO: n=8. (E) Body weight change of Con and Cacna1g POMC KO over the course 3 weeks feeding on P45 diet. (F-I) Feeding behaviour assessments of Con and Cacna1g POMC KO mice on P45 diets during indirect calorimetry measurement. ( F ) 24-hr profile of cumulative energy intake, ( G ) 24-hr total energy intake, ( H ) 24-hr average meal size and ( I ) 24-hr average meal number. (J-K) Energy expenditure of Con and Cacna1g POMC KO mice on P45 diets during indirect calorimetry measurement. ( J ) ANCOVA analysis of average 24hr energy expenditure against body weight of Con and Cacna1g MBH KO on P45 diets, ( K ) 24hr average energy expenditure. For (E-K), Con: n = 5; Cacna1g POMC KO : n = 7. *p<0.05, **p,0.01. Values are reported as mean ± SEM

Article Snippet: In brief, 33.3 μl beads per sample were washed with 1 mL TBS with 1% Triton X-100 (TBST) then blocked with 500 μl TBST with 1μg/ml BSA for 20 min at 4°C with rotation, followed by incubating with 1 μg N-terminal anti-Cav3.1 (Alomone Labs, Cat# ACC-021) (or 1 μg rabbit IgG as a control) in 500 μl lysis buffer with 1 μg/ml BSA at 4°C with rotation for 1 hr.

Techniques: Microscopy, CRISPR, Knock-Out, Expressing, Injection

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-B) Confocal i mmunofluorescence (IF) microscopy analysis of the CRISPR knockout efficiency of Cav3.1 in Cacna1g POMC KO mediobasal hypothalamus with a C-terminal specific Cav3.1 antibody validated in Fig. S3F. ( A ) Zoom-out images of . Scale bars: 200 µm. ( B ) Quantification of hypothalamic POMC neurons transduced with AAVs. Con: n = 5; Cacna1g POMC KO : n = 6. (C-E) Metabolic parameters of Cacna1g POMC KO mice on P45 diet. ( C ) 24 hr profile of energy expenditure, ( C ) 24 hr profiles of locomotor activity and ( D ) respiratory exchange ratio ( E ) during indirect calorimetry measurement. Con: n = 5; Cacna1g POMC KO : n = 7. (F) Oral glucose tolerance test of Con and Cacna1g MBH KO mice on P14 diet. Con: n = 5; Cacna1g POMC KO : n = 7. (G-I) Urine analyses of Con and Cacna1g POMC KO mice fed on P45 diets. Urinary albumin/creatinine ACR ratio ( G ), urea ( H ) and glucose ( I ). Con: n = 5; Cacna1g POMC KO : n = 7. (J) Acute feeding responses after MBH injection of Veh, 0.1 mM and 1 mM Ca v 3.1 activator SAK3. n = 14 per group. (K) Acute feeding responses after MBH injection of Veh, 0.1 mM SAK3, 1 µg/μl liraglutide (Lira) and SAK3+Lira co-treatment. n = 14 per group. (L-N) 24 hr feeding responses ( L ), 24 hr % body weight changes ( M ) and acute feeding responses ( N ) after MBH injection of Veh, 0.1 mM SAK3, 5ug/μl leptin (Lep) and SAK3+Lep co-treatment. n = 14 per group. (O-Q) 24 hr feeding responses ( O ), 24 hr % body weight changes ( P ) and acute feeding responses ( Q ) after MBH injection of Veh, 0.1 mM SAK3, 10ug/μl lorcaserin (Lor) and SAK3+Lor co-treatment. n = 14 per group. Groups denoted with different letter in (L, M, O, P) indicate significant difference (p<0.05). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Values are reported as mean ± SEM.

Article Snippet: In brief, 33.3 μl beads per sample were washed with 1 mL TBS with 1% Triton X-100 (TBST) then blocked with 500 μl TBST with 1μg/ml BSA for 20 min at 4°C with rotation, followed by incubating with 1 μg N-terminal anti-Cav3.1 (Alomone Labs, Cat# ACC-021) (or 1 μg rabbit IgG as a control) in 500 μl lysis buffer with 1 μg/ml BSA at 4°C with rotation for 1 hr.

Techniques: Microscopy, CRISPR, Knock-Out, Transduction, Activity Assay, Injection

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-B) 24-hr feeding responses ( A ) and % body weight changes ( B ) after MBH injection of Veh, 0.1 mM and 1 mM Cav3.1 activator SAK3. n = 14 per group. (C-F) Hypothalamic expression of Cav3.1 is required for the feeding effects of SAK3. 24-hr feeding responses and % body weight changes after MBH injection of Veh and 1mM SAK3 in Con (n = 6) ( C-D ) and Cacna1g MBH KO mice (n = 8) ( E-F ). (G-H) 24hr feeding responses ( G ) and % body weight changes ( H ) after MBH injection of Veh, 0.1 mM SAK3, 1 μg/μl liraglutide (Lira) and SAK3+Lira co-treatment. n = 14 per group. (I-J) Immunofluorescence (IF) microscopy analysis of POMC neuron activation after Lira and SAK3+Lira. ( I ) Representative fluorescence images of POMC (red) and cFos (green). White squares: regions shown in the zoom-in images. White arrows: neurons double positive for POMC and cFos IF. Scale bar: 200um. ( J ) Quantification of images analysis. Groups denoted with different letter in (A, B, G, H) indicate significant difference (p<0.05). *p<0.05, **p<0.01, ****p<0.0001. Values are reported as mean ± SEM.

Article Snippet: In brief, 33.3 μl beads per sample were washed with 1 mL TBS with 1% Triton X-100 (TBST) then blocked with 500 μl TBST with 1μg/ml BSA for 20 min at 4°C with rotation, followed by incubating with 1 μg N-terminal anti-Cav3.1 (Alomone Labs, Cat# ACC-021) (or 1 μg rabbit IgG as a control) in 500 μl lysis buffer with 1 μg/ml BSA at 4°C with rotation for 1 hr.

Techniques: Injection, Expressing, Immunofluorescence, Microscopy, Activation Assay, Fluorescence

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) Diagram of experimental strategy using intranasal administration (IN) of SAK3 to activate hypothalamic Cav3.1 activity. (B) 24 hours body weight change after IN administration of indicated substances on lean mice fed on normal chow. Comparisons were made against saline treated condition. Each treatment were 96 hours apart. n = 4 per repeated testing condition. (C-D) Mass spectrometry quantification of the tissue abundance of SAK3 in the MBH ( C ) and heart ( D ) 1 hour after IN administration. n = 1 for IN saline; n = 3 for IN SAK3. (E) Diagram of the experimental paradigm to test the weight loss effect of IN SAK3 in high fat diet induced obesity mouse model. n = 8 per group for (H-F). (F) Cumulative energy intake since the start of respective treatments over the course of 2 week treatment. (G-H) % Body weight change normalised to the start of respective treatments over the course of 2-week treatment ( G ). Histogram summarising the terminal % body weight change ( H ). For (F-G), on the top over the plots, colour-coded * indicates p<0.05 between respective Drug vs Veh treatment phases within the same group; at the bottom of the plots, ^p<0.05 SAK3 vs Lira; £ p<0.05 SAK vs SAK3+Lira; # p<0.05 Lira vs SAK3+Lira. *p<0.05, ****p<0.0001. Values are reported as mean ± SEM.

Article Snippet: In brief, 33.3 μl beads per sample were washed with 1 mL TBS with 1% Triton X-100 (TBST) then blocked with 500 μl TBST with 1μg/ml BSA for 20 min at 4°C with rotation, followed by incubating with 1 μg N-terminal anti-Cav3.1 (Alomone Labs, Cat# ACC-021) (or 1 μg rabbit IgG as a control) in 500 μl lysis buffer with 1 μg/ml BSA at 4°C with rotation for 1 hr.

Techniques: Activity Assay, Saline, Mass Spectrometry