rabbit  (Alomone Labs)

Journal: International Journal of Molecular Sciences

Article Title: Impact of C-Terminal PKC Phosphorylation on TRPC6 Current Kinetics

doi: 10.3390/ijms262311482

Figure Lengend Snippet: Amino acid exchanges of serine 768 result in distinct current kinetics. Electrophysiological whole-cell measurements of HEK293T cells overexpressing TRPC6 and indicated TRPC6 mutants in the presence of 10 µM OptoBI-1 ( A – H ) or 30 µM OptoDArG ( I – P ). ( A , I ) Summaries of current densities (‘Curr. dens.’) at potentials of ±100 mV evoked by light. First small boxplots represent current densities in the presence of blue light (442 nm) which establishes trans -configuration and second boxplots represent maximal current densities in the presence of UV light (367 nm) which establishes cis -configuration. ( A ) No significant differences were observed between trans- OptoBI-1-induced or cis -OptoBI-1-induced current densities of the mutant channels compared to the wildtype. ( I ) trans -OptoDArG- or cis -OptoDArG-induced current densities of the mutant channels were not significantly different compared to the wildtype. ( B , J ) Representative current density–voltage relations induced by illumination with UV light. ( C , F , K , N ) Representative normalized current time courses of inward currents at constant holding potential of -60 mV during photoswitching from blue light (blue bar) to UV light (magenta bar) ( F , N ) and back to blue light (blue bar) ( C , K ). ( D , E , L , M ) Summaries of half-life time constants (τ H ) of the activation ( D , L ) and deactivation ( E , M ) kinetics. ( G , H , O , P ) Summaries of half-life time constants (τ H ) of the fast ( G , O ) and slow ( H , P ) inactivation kinetics. ( D , E , G , H , L , M , O , P ) Data are displayed as boxplots and interquartile ranges. Numbers over boxplots indicate number of measured cells. Statistical significance was determined using the Kruskal–Wallis test with Dunn’s post hoc analysis. Gray asterisks indicate overall differences among all groups identified by the Kruskal–Wallis test, whereas colored and black asterisks indicate pairwise differences revealed by Dunn’s post hoc analysis (colored vs. wild type; black between the groups connected by horizontal lines). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Afterwards, rabbit anti-TRPC6 antibody (Proteintech, Planegg-Martinsried, Germany; Cat. No.: 18236-1-AP) was diluted 1:1000 in TBS-T containing 5% BSA and the blot was incubated for 12–18 h at 4 °C.

Techniques: Mutagenesis, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Impact of C-Terminal PKC Phosphorylation on TRPC6 Current Kinetics

doi: 10.3390/ijms262311482

Figure Lengend Snippet: Amino acid exchanges of serine 814 influence the current kinetics. Electrophysiological whole-cell measurements of HEK293T cells overexpressing TRPC6 and indicated TRPC6 mutants in the presence of 10 µM OptoBI-1 ( A – H ) or 30 µM OptoDArG ( I – P ). ( A , I ) Summaries of current densities (‘Curr. dens.’) at potentials of ±100 mV evoked by light. First small boxplots represent current densities in the presence of blue light which establishes trans -configuration, and second boxplots represent maximal current densities in the presence of UV light which establishes cis -configuration. ( A ) No significant differences were observed between trans- OptoBI-1-induced or cis -OptoBI-1-induced current densities of the mutant channels compared to the wildtype. ( I ) trans -OptoDArG- or cis -OptoDArG-induced current densities of the mutant channels were not significantly different compared to the wildtype. ( B , J ) Representative current-density–voltage relations induced by illumination with UV light. ( C , F , K , N ) Representative normalized current time courses of inward currents at constant holding potential of −60 mV during photoswitching from blue light (blue bar) to UV light (magenta bar) ( F , N ) and back to blue light (blue bar) ( C , K ). ( D , E , L , M ) Summaries of half-life time constants (τ H ) of the activation ( D , L ) and deactivation ( E , M ) kinetics. ( G , H , O , P ) Summaries of half-life time constants (τ H ) of the fast ( G , O ) and slow ( H , P ) inactivation kinetics. ( D , E , G , H , L , M , O , P ) Data are displayed as boxplots and interquartile ranges. Numbers over boxplots indicate number of measured cells. Gray asterisks indicate overall differences among all groups identified by the Kruskal–Wallis test, whereas colored and black asterisks indicate pairwise differences revealed by Dunn’s post hoc analysis (colored vs. wild type; black between the groups connected by horizontal lines). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Afterwards, rabbit anti-TRPC6 antibody (Proteintech, Planegg-Martinsried, Germany; Cat. No.: 18236-1-AP) was diluted 1:1000 in TBS-T containing 5% BSA and the blot was incubated for 12–18 h at 4 °C.

Techniques: Mutagenesis, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Impact of C-Terminal PKC Phosphorylation on TRPC6 Current Kinetics

doi: 10.3390/ijms262311482

Figure Lengend Snippet: Amino acid exchanges of serine 835 alter the current kinetics. Electrophysiological whole-cell measurements of HEK293T cells overexpressing TRPC6 and indicated TRPC6 mutants in the presence of 10 µM OptoBI-1 ( A – H ) or 30 µM OptoDArG ( I – P ). ( A , I ) Summaries of current densities (‘Curr. dens.’) at potentials of ±100 mV evoked by light. First small boxplots represent current densities in the presence of blue light) which establishes trans -configuration and second boxplots represent maximal current densities in the presence of UV light which establishes cis -configuration. ( A ) No significant differences were observed between trans- OptoBI-1-induced or cis -OptoBI-1-induced current densities of the mutant channels compared to the wildtype. ( I ) trans -OptoDArG- or cis -OptoDArG-induced current densities of the mutant channels were not significantly different compared to the wildtype. ( B , J ) Representative current-density–voltage relations induced by illumination with UV light. ( C , F , K , N ) Representative normalized current time courses of inward currents at constant holding potential of -60 mV during photoswitching from blue light (blue bar) to UV light (magenta bar) ( F , N ) and back to blue light (blue bar) ( C , K ). ( D , E , L , M ) Summaries of half-life time constants (τ H ) of the activation ( D , L ) and deactivation ( E , M ) kinetics. ( G , H , O , P ) Summaries of half-life time constants (τ H ) of the fast ( G , O ) and slow ( H , P ) inactivation kinetics. ( D , E , G , H , L , M , O , P ) Data are displayed as boxplots and interquartile ranges. Numbers over boxplots indicate number of measured cells. Gray asterisks indicate overall differences among all groups identified by the Kruskal–Wallis test, whereas colored and black asterisks indicate pairwise differences revealed by Dunn’s post hoc analysis (colored vs. wild type; black between the groups connected by horizontal lines). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Afterwards, rabbit anti-TRPC6 antibody (Proteintech, Planegg-Martinsried, Germany; Cat. No.: 18236-1-AP) was diluted 1:1000 in TBS-T containing 5% BSA and the blot was incubated for 12–18 h at 4 °C.

Techniques: Mutagenesis, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Impact of C-Terminal PKC Phosphorylation on TRPC6 Current Kinetics

doi: 10.3390/ijms262311482

Figure Lengend Snippet: Amino acid exchanges of serine 892 influence current densities and current kinetics. Electrophysiological whole-cell measurements of HEK293T cells overexpressing TRPC6 and indicated TRPC6 mutants in the presence of 10 µM OptoBI-1 ( A – H ) or 30 µM OptoDArG ( I – P ). ( A , I ) Summaries of current densities (‘Curr. dens.’) at potentials of ±100 mV evoked by light. First small boxplots represent current densities in the presence of blue light which establishes trans -configuration, and second boxplots represent maximal current densities in the presence of UV light which establishes cis -configuration. ( A ) Significant differences were observed between cis- OptoBI-1-induced current densities of the mutants S892A and S892D compared to the wildtype. ( I ) cis -OptoDArG-induced current densities of the mutants S892D and S892E were significantly different compared to the wildtype. ( B , J ) Representative current-density–voltage relations induced by illumination with UV light. ( C , F , K , N ) Representative normalized current time courses of inward currents at constant holding potential of −60 mV during photoswitching from blue light (blue bar) to UV light (magenta bar) ( F , N ) and back to blue light (blue bar) ( C , K ). ( D , E , L , M ) Summaries of half-life time constants (τ H ) of the activation ( D , L ) and deactivation ( E , M ) kinetics. ( G , H , O , P ) Summaries of half-life time constants (τ H ) of the fast ( G , O ) and slow ( H , P ) inactivation kinetics. ( D , E , G , H , L , M , O , P ) Data are displayed as boxplots and interquartile ranges. Numbers over boxplots indicate number of measured cells. Gray asterisks indicate overall differences among all groups identified by the Kruskal–Wallis test, whereas colored and black asterisks indicate pairwise differences revealed by Dunn’s post hoc analysis (colored vs. wild type; black between the groups connected by horizontal lines). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Afterwards, rabbit anti-TRPC6 antibody (Proteintech, Planegg-Martinsried, Germany; Cat. No.: 18236-1-AP) was diluted 1:1000 in TBS-T containing 5% BSA and the blot was incubated for 12–18 h at 4 °C.

Techniques: Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Impact of C-Terminal PKC Phosphorylation on TRPC6 Current Kinetics

doi: 10.3390/ijms262311482

Figure Lengend Snippet: Amino acid exchanges of serine 928 cause changes in the current kinetics. Electrophysiological whole-cell measurements of HEK293T cells overexpressing TRPC6 and indicated TRPC6 mutants in the presence of 10 µM OptoBI-1 ( A – H ) or 30 µM OptoDArG ( I – P ). ( A . I ) Summaries of current densities (‘Curr. dens.’) at potentials of ±100 mV evoked by light. First small boxplots represent current densities in the presence of blue light, which establishes trans -configuration, and second boxplots represent maximal current densities in the presence of UV light, which establishes cis -configuration. ( A ) No significant differences were observed between trans- OptoBI-1-induced or cis -OptoBI-1-induced current densities of the mutant channels compared to the wildtype. ( I ) trans -OptoDArG- or cis -OptoDArG-induced current densities of the mutant channels were not significantly different compared to the wildtype. ( B , J ) Representative current-density–voltage relations induced by illumination with UV light. ( C , F , K , N ) Representative normalized current time courses of inward currents at constant holding potential of -60 mV during photoswitching from blue light (blue bar) to UV light (magenta bar) ( F , N ) and back to blue light (blue bar) ( C , K ). ( D , E , L , M ) Summaries of half-life time constants (τ H ) of the activation ( D , L ) and deactivation ( E , M ) kinetics. ( G , H , O , P ) Summaries of half-life time constants (τ H ) of the fast ( G , O ) and slow ( H , P ) inactivation kinetics. ( D , E , G , H , L , M , O , P ) Data are displayed as boxplots and interquartile ranges. Numbers over boxplots indicate number of measured cells. Gray asterisks indicate overall differences among all groups identified by the Kruskal–Wallis test, whereas colored and black asterisks indicate pairwise differences revealed by Dunn’s post hoc analysis (colored vs. wild type; black between the groups connected by horizontal lines). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Afterwards, rabbit anti-TRPC6 antibody (Proteintech, Planegg-Martinsried, Germany; Cat. No.: 18236-1-AP) was diluted 1:1000 in TBS-T containing 5% BSA and the blot was incubated for 12–18 h at 4 °C.

Techniques: Mutagenesis, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Impact of C-Terminal PKC Phosphorylation on TRPC6 Current Kinetics

doi: 10.3390/ijms262311482

Figure Lengend Snippet: Amino acid exchange from serine 928 to glycine and C-terminal truncations influence the current kinetics. Electrophysiological whole-cell measurements of HEK293T cells overexpressing TRPC6 and indicated TRPC6 mutants in the presence of 10 µM OptoBI-1 ( A – H ) or 30 µM OptoDArG ( I – P ). ( A , I ) Summaries of current densities (‘Curr. dens.’) at potentials of ±100 mV evoked by light. First small boxplots represent current densities in the presence of blue light, which establishes trans -configuration, and second boxplots represent maximal current densities in the presence of UV light, which establishes cis -configuration. ( A ) No significant differences were observed between trans- OptoBI-1-induced or cis -OptoBI-1-induced current densities of the mutant channels compared to the wildtype. ( I ) trans -OptoDArG- or cis -OptoDArG-induced current densities of the mutant channels were not significantly different compared to the wildtype. ( B , J ) Representative current-density–voltage relations induced by illumination with UV light. ( C , F , K , N ) Representative normalized current time courses of inward currents at constant holding potential of -60 mV during photoswitching from blue light (blue bar) to UV light (magenta bar) ( F , N ) and back to blue light (blue bar) ( C , K ). ( D , E , L , M ) Summaries of half-life time constants (τ H ) of the activation ( D , L ) and deactivation ( E , M ) kinetics. ( G , H , O , P ) Summaries of half-life time constants (τ H ) of the fast ( G , O ) and slow ( H , P ) inactivation kinetics. ( D , E , G , H , L , M , O , P ) Data are displayed as boxplots and interquartile ranges. Numbers over boxplots indicate number of measured cells. Gray asterisks indicate overall differences among all groups identified by the Kruskal–Wallis test, whereas colored and black asterisks indicate pairwise differences revealed by Dunn’s post hoc analysis (colored vs. wild type; black between the groups connected by horizontal lines). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Afterwards, rabbit anti-TRPC6 antibody (Proteintech, Planegg-Martinsried, Germany; Cat. No.: 18236-1-AP) was diluted 1:1000 in TBS-T containing 5% BSA and the blot was incubated for 12–18 h at 4 °C.

Techniques: Mutagenesis, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Impact of C-Terminal PKC Phosphorylation on TRPC6 Current Kinetics

doi: 10.3390/ijms262311482

Figure Lengend Snippet: Multiple amino acid exchanges influence the current kinetics. Electrophysiological whole-cell measurements of HEK293T cells overexpressing TRPC6 or TRPC6 double (S814A and S835A), quadruple (S768A, S814A, S835A, and S892A), or quintuple (S768A, S814A, S835A, S892A, and S928A) mutants in the presence of 10 µM OptoBI-1 ( A – H ) or 30 µM OptoDArG ( I – P ). ( A , I ) Summaries of current densities (‘Curr. dens.’) at potentials of ±100 mV evoked by light. First small boxplots represent current densities in the presence of blue light, which establishes trans -configuration, and second boxplots represent maximal current densities in the presence of UV light, which establishes cis -configuration. ( A ) No significant differences were observed between trans- OptoBI-1-induced or cis -OptoBI-1-induced current densities of the mutant channels compared to the wildtype. ( I ) trans -OptoDArG- or cis -OptoDArG-induced current densities of the mutant channels were not significantly different compared to the wildtype. ( B , J ) Representative current-density–voltage relations induced by illumination with UV light. ( C , F , K , N ) Representative normalized current time courses of inward currents at constant holding potential of -60 mV during photoswitching from blue light (blue bar) to UV light (magenta bar) ( F , N ) and back to blue light (blue bar) ( C , K ). ( D , E , L , M ) Summaries of half-life time constants (τ H ) of the activation ( D , L ) and deactivation ( E , M ) kinetics. ( G , H , O , P ) Summaries of half-life time constants (τ H ) of the fast ( G , O ) and slow ( H , P ) inactivation kinetics. ( D , E , G , H , L , M , O , P ) Data are displayed as boxplots and interquartile ranges. Numbers over boxplots indicate number of measured cells. Gray asterisks indicate overall differences among all groups identified by the Kruskal–Wallis test, whereas colored and black asterisks indicate pairwise differences revealed by Dunn’s post hoc analysis (colored vs. wild type; black between the groups connected by horizontal lines). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Afterwards, rabbit anti-TRPC6 antibody (Proteintech, Planegg-Martinsried, Germany; Cat. No.: 18236-1-AP) was diluted 1:1000 in TBS-T containing 5% BSA and the blot was incubated for 12–18 h at 4 °C.

Techniques: Mutagenesis, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Impact of C-Terminal PKC Phosphorylation on TRPC6 Current Kinetics

doi: 10.3390/ijms262311482

Figure Lengend Snippet: PKC phosphorylation and dephosphorylation alter the current kinetics. Electrophysiological whole-cell measurements of HEK293T cells overexpressing TRPC6 incubated with the PKC activator PMA (1 µM) or with the PKC inhibitors BIM I (1 µM) or ceramide (2 µM) for 20 min at room temperature in the presence of 10 µM OptoBI-1 ( A – H ) or 30 µM OptoDArG ( I – P ). ( A , I ) Summaries of current densities (‘Curr. dens.’) at potentials of ±100 mV evoked by light. First small boxplots represent current densities in the presence of blue light, which establishes trans -configuration, and second boxplots represent maximal current densities in the presence of UV light, which establishes cis -configuration. Significant differences were observed between cis -OptoBI-1-induced current densities in the presence of PMA compared to the wildtype. ( B , J ) Representative current-density–voltage relations induced by illumination with UV light. ( C , F , K , N ) Representative normalized current time courses of inward currents at constant holding potential of -60 mV during photoswitching from blue light (blue bar) to UV light (magenta bar) ( F , N ) and back to blue light (blue bar) ( C , K ). ( D , E , L , M ) Summaries of half-life time constants (τ H ) of the activation ( D , L ) and deactivation ( E , M ) kinetics. ( G , H , O , P ) Summaries of half-life time constants (τ H ) of the fast ( G , O ) and slow ( H , P ) inactivation kinetics. ( D , E , G , H , L , M , O , P ) Data are displayed as boxplots and interquartile ranges. Numbers over boxplots indicate number of measured cells. Gray asterisks indicate overall differences among all groups identified by the Kruskal–Wallis test, whereas colored and black asterisks indicate pairwise differences revealed by Dunn’s post hoc analysis (colored vs. wild type; black between the groups connected by horizontal lines). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Afterwards, rabbit anti-TRPC6 antibody (Proteintech, Planegg-Martinsried, Germany; Cat. No.: 18236-1-AP) was diluted 1:1000 in TBS-T containing 5% BSA and the blot was incubated for 12–18 h at 4 °C.

Techniques: Phospho-proteomics, De-Phosphorylation Assay, Incubation, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Impact of C-Terminal PKC Phosphorylation on TRPC6 Current Kinetics

doi: 10.3390/ijms262311482

Figure Lengend Snippet: The quadruple mutant incubated with ceramide behaves like the quintuple mutant. Electrophysiological whole-cell measurements of HEK293T cells overexpressing TRPC6 or the TRPC6 quadruple (S768A, S814A, S835A and S892A) mutant, the quadruple mutant incubated with the PKC inhibitor ceramide (2 µM for 20 min at room temperature), or the quintuple (S768A, S814A, S835A, S892A and S928A) mutant in the presence of 10 µM OptoBI-1 ( A – H ) or 30 µM OptoDArG ( I – P ). ( A , I ) Summaries of current densities (‘Curr. dens.’) at potentials of ±100 mV evoked by light. First small boxplots represent current densities in the presence of blue light, which establishes trans -configuration, and second boxplots represent maximal current densities in the presence of UV light, which establishes cis -configuration. ( A ) No significant differences were observed between trans- OptoBI-1-induced or cis -OptoBI-1-induced current densities of the mutant channels compared to the wildtype. ( I ) trans -OptoDArG- or cis -OptoDArG-induced current densities of the mutant channels were not significantly different compared to the wildtype. ( B , J ) Representative current-density–voltage relations induced by illumination with UV light. ( C , F , K , N ) Representative normalized current time courses of inward currents at constant holding potential of -60 mV during photoswitching from blue light (blue bar) to UV light (magenta bar) ( F , N ) and back to blue light (blue bar) ( C , K ). ( D , E , L , M ) Summaries of half-life time constants (τ H ) of the activation ( D , L ) and deactivation ( E , M ) kinetics. ( G , H , O , P ) Summaries of half-life time constants (τ H ) of the fast ( G , O ) and slow ( H , P ) inactivation kinetics. ( D , E , G , H , L , M , O , P ) Data are displayed as boxplots and interquartile ranges. Numbers over boxplots indicate number of measured cells. Gray asterisks indicate overall differences among all groups identified by the Kruskal–Wallis test, whereas colored and black asterisks indicate pairwise differences revealed by Dunn’s post hoc analysis (colored vs. wild type; black between the groups connected by horizontal lines). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Afterwards, rabbit anti-TRPC6 antibody (Proteintech, Planegg-Martinsried, Germany; Cat. No.: 18236-1-AP) was diluted 1:1000 in TBS-T containing 5% BSA and the blot was incubated for 12–18 h at 4 °C.

Techniques: Mutagenesis, Incubation, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Impact of C-Terminal PKC Phosphorylation on TRPC6 Current Kinetics

doi: 10.3390/ijms262311482

Figure Lengend Snippet: Additional PKC activation did not alter the current kinetics of the quintuple mutant in electrophysiological whole-cell measurements of HEK293T cells overexpressing TRPC6, the TRPC6 quintuple (S768A, S814A, S835A, S892A, and S928A) mutant, or the quintuple mutant incubated with the PKC activator PMA (1 µM for 20 min at room temperature) in the presence of 10 µM OptoBI-1 ( A – H ) or 30 µM OptoDArG ( I – P ). ( A , I ) Summaries of current densities (‘Curr. dens.’) at potentials of ±100 mV evoked by light. First small boxplots represent current densities in the presence of blue light, which establishes trans -configuration, and second boxplots represent maximal current densities in the presence of UV light, which establishes cis -configuration. Significant differences between cis -OptoBI-1-induced current densities of the quintuple mutant incubated with PMA compared to the wildtype channel were observed. ( B , J ) Representative current-density–voltage relations induced by illumination with UV light. ( C , F , K , N ) Representative normalized current time courses of inward currents at constant holding potential of -60 mV during photoswitching from blue light (blue bar) to UV light (magenta bar) ( F , N ) and back to blue light (blue bar) ( C , K ). ( D , E , L , M ) Summaries of half-life time constants (τ H ) of the activation ( D , L ) and deactivation ( E , M ) kinetics. ( G , H , O , P ) Summaries of half-life time constants (τ H ) of the fast ( G , O ) and slow ( H , P ) inactivation kinetics. ( D , E , G , H , L , M , O , P ) Data are displayed as boxplots and interquartile ranges. Numbers over boxplots indicate number of measured cells. Gray asterisks indicate overall differences among all groups identified by the Kruskal–Wallis test, whereas colored and black asterisks indicate pairwise differences revealed by Dunn’s post hoc analysis (colored vs. wild type; black between the groups connected by horizontal lines). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Afterwards, rabbit anti-TRPC6 antibody (Proteintech, Planegg-Martinsried, Germany; Cat. No.: 18236-1-AP) was diluted 1:1000 in TBS-T containing 5% BSA and the blot was incubated for 12–18 h at 4 °C.

Techniques: Activation Assay, Mutagenesis, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Impact of C-Terminal PKC Phosphorylation on TRPC6 Current Kinetics

doi: 10.3390/ijms262311482

Figure Lengend Snippet: PKC inhibition alters current kinetics and normalized slope conductance of endogenously expressed TRPC6 channels. ( A ) Western blot analysis of HEK293T cells, HEK293T cells overexpressing human TRPC6, and of human renal proximal tubule endothelial cells (RPTEC) endogenously expressing low amounts of TRPC6. The red box shows human TRPC6 expression at 106 kDa. The amount of protein used, and the different exposure times are indicated on the top or, respectively, to the right of the images. ( B – E ) Electrophysiological whole-cell measurements of RPTEC and of RPTEC incubated with ceramide (2 µM for 20 min at room temperature) in the presence of 30 µM OptoDArG. ( B ) Summary of current densities (‘Curr. dens.’) at potentials of ±100 mV evoked by light. First small boxplots represent current densities in the presence of blue light, which establishes trans -configuration, and second boxplots represent maximal current densities in the presence of UV light, which establishes cis -configuration. Significant differences between cis -OptoDArG-induced current densities of RPTEC in the presence or absence of ceramide were observed (* p < 0.05, ** p < 0.01; Mann–Whitney U test). ( C , D ) Summaries of half-life time constants (τ H ) of the activation ( C ) and deactivation ( D ) kinetics (* p < 0.05, *** p < 0.001; Mann–Whitney U test). ( B – D ) Data are displayed as boxplots and interquartile ranges. Numbers over boxplots indicate number of measured cells. ( E ) Current-density–voltage relations (‘Curr. dens.’) of cis -OptoDArG-induced currents in the presence or absence of ceramide are displayed (above). The current-density–voltage relations were separately smoothed and normalized (‘Curr. dens norm (%)’) at negative and positive potentials. The calculated normalized slope conductance (NSC) (‘Norm. G slope ’) is displayed as mean ± SD. p values are calculated using Mann–Whitney U-test.

Article Snippet: Afterwards, rabbit anti-TRPC6 antibody (Proteintech, Planegg-Martinsried, Germany; Cat. No.: 18236-1-AP) was diluted 1:1000 in TBS-T containing 5% BSA and the blot was incubated for 12–18 h at 4 °C.

Techniques: Inhibition, Western Blot, Expressing, Incubation, MANN-WHITNEY, Activation Assay

Journal: Cell reports

Article Title: Inducing ferroptosis to impede metastasis by inhibiting the calcium channel TRPC6

doi: 10.1016/j.celrep.2025.116543

Figure Lengend Snippet: (A) Relative viability of p27 + and p27 − sorted p27-mVenus CAL-51 cells after 24 h treatment with either DMSO or IKE in the presence or absence of ferrostatin-1, measured using the CellTiter-Glo (CTG) assay. (B) Expression of TRPC6 in p27 + and p27 − CAL-51 cells, assessed by immunoblotting. (C) p27 + CAL-51 cells were treated for 24 h with either DMSO or increasing concentrations of IKE in the presence of either BI-749327 (10 μM) or ferrostatin-1 (2 μM), or a combination of BI-749327 and ferrostatin-1. Viability was measured using the CTG assay. (D) Mice injected with either CAL-51 p27 − or CAL-51 p27 + cells were sacrificed 6 weeks post tail-vein injection, and metastases in the lung were assessed by H&E staining. The percentage of metastasis was quantified and is presented. (E) Expression of TRPC6 mRNA in primary tumor and circulating tumor cells (CTCs) in breast cancer patients obtained from ctcRbase. (F) Relative viability of LM2 cells after treatment with DMSO, IKE (1 μM), ferrostatin-1 (2 μM), or their combination for 24 h. (G) Relative viability of parental LM2 cells (Par) and an LM2 IKE-resistant (IKE-Res) sub-population after 48 h, measured using the CTG assay. (H) Quantification of p27 mRNA by qPCR in LM2 parental and IKE-Res cells. (I) Quantification of TRPC6 mRNA by qPCR in LM2 parental and IKE-Res cells. (J) Time course of Ca 2+ flux measured by mean fluorescence intensity following CaCl 2 exposure in LM2 parental and LM2 IKE-Res cells pretreated with Fluo4-AM. (K) Relative viability of LM2 parental cells and LM2 cells that had been transfected with a TRPC6 expression plasmid (TRPC6-WT) and then treated with increasing IKE concentrations for 24 h, measured using the CTG assay. (L) Quantification of p27 mRNA by qPCR in LM2 parental and LM2 TRPC6-WT-overexpressing cells. The bars in graphs represent mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Rabbit polyclonal Anti-TRPC6 Antibody , Alomone Labs , Cat#ACC-017; RRID: AB_2040243.

Techniques: CTG Assay, Expressing, Western Blot, Injection, Staining, Fluorescence, Transfection, Plasmid Preparation

Journal: Cell reports

Article Title: Inducing ferroptosis to impede metastasis by inhibiting the calcium channel TRPC6

doi: 10.1016/j.celrep.2025.116543

Figure Lengend Snippet: (A) The level of GSH was quantified in p27 + and p27 − sorted p27-mVenus CAL-51 cells. (B) The level of GSH levels was quantified in p27 + CAL-51 cells that have been pretreated with either BI-749327 (10 μM) or DMSO for 48 h and subsequently treated with IKE (10 μM) or DMSO for 24 h. (C) Expression of glutamate-cysteine ligase catalytic (GCLC) subunit mRNA in LM2 (parental) and LM2 IKE-resistant (IKE-Res) cells was quantified by qPCR. (D) GSEA of transcriptomic data from p27 + and p27 − CAL-51 cells shows enrichment of c-Myc targets in p27 − cells. (E) Expression of c-Myc in p27 + and p27 − CAL-51 cells and in LM2 parental and LM2 IKE-Res cells shows enrichment of c-Myc targets in p27 − CAL-51 and LM2 cells, assessed by immunoblotting. (F) Expression of c-Myc mRNA in primary tumor and circulating tumor cells (CTCs) in breast cancer patients obtained from ctcRbase. (G) mRNA expression of TRPC6 , c-Myc , CDC25a , and GCLC was quantified in p27 + CAL-51 cells that had been treated with siTRPC6 or siCTRL for 24 h. (H) The level of GSH was quantified after 30 min in suspension in CAL-51 p27 + cells treated with siTRPC6 or siCTRL for 24 h. (I) mRNA expression of TRPC6 , c-Myc , and GCLC was quantified in CAL-51 p27 − parental or CAL-51 p27 − TRPC6-overexpressing (TRPC6-WT) cells. (J) The concentration of GSH was quantified after 30 min in suspension in CAL-51 p27 − parental and TRPC6-WT cells. (K) Relative viability of p27 − CAL-51 cells that had been treated with increasing concentrations of IKE in the presence of either DMSO or 10074-G5 (50 μM) for 24 h. (L) Relative viability of LM2 cells that had been treated with increasing concentrations of IKE in the presence of either DMSO or 10074-G5 (50 μM) for 24 h. (M) The concentration of GSH was quantified in p27 − CAL-51 cells after 24-h treatment with either 10074-G5 (50 μM) or DMSO. (N) The level of GSH was quantified in LM2 cells after 24-h treatment with either 10074-G5 (50 μM) or DMSO. (O) GCLC mRNA expression was quantified by qPCR in LM2 cells that had been treated with 10074-G5 (50 μM) or DMSO for 24 h. The bars in graphs represent mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Rabbit polyclonal Anti-TRPC6 Antibody , Alomone Labs , Cat#ACC-017; RRID: AB_2040243.

Techniques: Expressing, Western Blot, Suspension, Concentration Assay

Journal: Cell reports

Article Title: Inducing ferroptosis to impede metastasis by inhibiting the calcium channel TRPC6

doi: 10.1016/j.celrep.2025.116543

Figure Lengend Snippet: (A) Total ROS measured by DCF fluorescence of p27 + and p27 − CAL-51 cells 30 min after matrix detachment. (B) Relative viability of p27 + and p27 − CAL-51 cells 48 h after matrix detachment as measured by trypan blue exclusion. (C) Total ROS measured by DCF fluorescence in p27 − CAL-51 cells after pretreatment with 10074-G5 (50 μM) or DMSO for 24 h and matrix detachment for 30 min (D) The level of GSH was quantified in p27 − CAL-51 cells after pretreatment with 10074-G5 (50 μM) or DMSO for 24 h and matrix detachment for 30 min (E) Relative viability of p27 + CAL-51 cells assessed by trypan blue exclusion after pretreatment with BI-749327 (10 μM) or DMSO for 24 h and matrix detachment for 48 h in the presence of either DMSO or ferrostatin-1 (2 μM). (F) Relative viability of p27 − CAL-51 cells that were engineered to express TRPC6 (TRPC6-WT) after pretreatment with BI-749327 (10 μM) or DMSO for 24 h and matrix detachment for 48 h in the presence of either DMSO or ferrostatin-1 (2 μM). (G) Relative viability of IKE-Res, TRPC6-expressing (TRPC6-WT), and parental LM2 cells pretreated with either BI-749327 (10 μM) or DMSO for 24 h before matrix detachment for 48 h. (H) Relative viability of LM2 IKE-Res and LM2 IKE-Res cells that were engineered to express c-Myc after matrix detachment for 48 h. (I) GSH was quantified in LM2 cells pretreated with 10074-G5 (50 μM) or DMSO for 24 h before matrix detachment for 30 min (J) Total ROS measured by DCF fluorescence of LM2 cells pretreated with either 10074-G5 (50 μM) or DMSO for 24 h before matrix detachment for 30 min (K) Relative viability assay of LM2 cells pretreated with 10074-G5 (50 μM) or DMSO for 24 h before matrix detachment for 48 h as assessed by trypan blue exclusion. The bars in graphs represent mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Rabbit polyclonal Anti-TRPC6 Antibody , Alomone Labs , Cat#ACC-017; RRID: AB_2040243.

Techniques: Fluorescence, Expressing, Viability Assay

Journal: International Journal of Molecular Sciences

Article Title: TRPC6 Deficiency Attenuates Mitochondrial and Cardiac Dysfunction in Heart Failure with Preserved Ejection Fraction Induced by High-Fat Diet Plus L-NAME

doi: 10.3390/ijms26199383

Figure Lengend Snippet: Cardiac systolic and diastolic functions assessed by echocardiography in WT and TRPC6 KO. ( A ) Stroke volume, ( B ) cardiac output, ( C ) ejection fraction, ( D ) fractional shortening, ( E ) isovolumic relaxation time, ( F ) myocardial performance index, and ( G ) E/e’, measurements at baseline, 4 weeks, and 8 weeks after L-NAME. Results are expressed as mean ± SEM, n = 6–7, * p < 0.05 between two groups by post hoc test after two-way ANOVA. Bar colors indicate experimental groups: white = WT RD (regular diet), gray = WT HFD+L-NAME, light blue = KO RD, dark blue = KO HFD+L-NAME.

Article Snippet: After transfer to nitrocellulose membranes, blots were rinsed in PBS and blocked in Odyssey blocking buffer (LI-CORbio, Lincoln, NE, USA) for 1 h at room temperature and then incubated with TRPC6 rabbit antibody (1:500, ACC-120, Alomone Labs, Jerusalem, Israel) or TRPC3 rabbit antibody (1:500, #77934, Cell Signaling Technology, Danvers, MA, USA), VDAC rabbit antibody (1:1000, #4661, Cell Signaling), GAPDH rabbit antibody (1:2000, #2118, Cell Signaling), caspase 9 rabbit antibody (1:1000, #9504, Cell Signaling), cleaved caspase 9 rabbit antibody (1:1000, #7237, Cell Signaling), PGC-1α rabbit antibody (1:1000, #2178, Cell Signaling) and BNP rabbit antibody (1:1000, PA5-96084, ThermoFisher, Waltham, MA, USA) at 4 °C overnight.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: TRPC6 Deficiency Attenuates Mitochondrial and Cardiac Dysfunction in Heart Failure with Preserved Ejection Fraction Induced by High-Fat Diet Plus L-NAME

doi: 10.3390/ijms26199383

Figure Lengend Snippet: Left ventricle wall thickness and fibrosis in WT and TRPC6 KO mice. ( A ) End-systolic left ventricular anterior wall thickness, ( B ) end-diastolic left ventricular anterior wall thickness, ( C ) end-systolic left ventricular posterior wall thickness, and ( D ) end-diastolic left ventricular posterior wall thickness, with measurements at baseline, 4 weeks and 8 weeks after L-NAME. Results are expressed as mean ± SEM, n = 6–8, * p < 0.05 between two groups by post hoc test after two-way ANOVA. Myocardial fibrosis measurements in WT and TRPC6 KO mice. Representative Masson Trichrome staining of left ventricles from WT RD ( E ), WT HFD+ L-NAME ( F ), KO RD ( G ), and KO HFD+ L-NAME ( H ). ( I ) Quantitative analysis of collagen fractions (blue color) by Masson Trichrome staining in the left ventricles. Results are expressed as mean ± SEM, n = 6, * p < 0.05 between two groups by non-parametric test after one-way ANOVA. ns, not significant. Bar colors indicate experimental groups: white = WT RD (regular diet), gray = WT HFD+L-NAME, light blue = KO RD, dark blue = KO HFD+L-NAME.

Article Snippet: After transfer to nitrocellulose membranes, blots were rinsed in PBS and blocked in Odyssey blocking buffer (LI-CORbio, Lincoln, NE, USA) for 1 h at room temperature and then incubated with TRPC6 rabbit antibody (1:500, ACC-120, Alomone Labs, Jerusalem, Israel) or TRPC3 rabbit antibody (1:500, #77934, Cell Signaling Technology, Danvers, MA, USA), VDAC rabbit antibody (1:1000, #4661, Cell Signaling), GAPDH rabbit antibody (1:2000, #2118, Cell Signaling), caspase 9 rabbit antibody (1:1000, #9504, Cell Signaling), cleaved caspase 9 rabbit antibody (1:1000, #7237, Cell Signaling), PGC-1α rabbit antibody (1:1000, #2178, Cell Signaling) and BNP rabbit antibody (1:1000, PA5-96084, ThermoFisher, Waltham, MA, USA) at 4 °C overnight.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: TRPC6 Deficiency Attenuates Mitochondrial and Cardiac Dysfunction in Heart Failure with Preserved Ejection Fraction Induced by High-Fat Diet Plus L-NAME

doi: 10.3390/ijms26199383

Figure Lengend Snippet: Echocardiography during dobutamine stress test. Time-dependent changes after dobutamine injection in ( A ) Heart rate, ( B ) ejection fraction, ( C ) stroke volume, ( D ) cardiac output, ( E ) LVESV, and ( F ) LVEDV in WT and TRPC6 KO mice with RD. ( G – L ) Heart rate, ejection fraction, stroke volume, cardiac output, LVESV, and LVEDV in WT and TRPC6 KO mice with HFD+L-NAME after dobutamine injection. Results are expressed as mean ± SEM, n = 5–6, *, p < 0.05 when compared with baseline in WT mice, #, p < 0.05 when compared to baseline in TRPC6 KO mice by post hoc test after one-way ANOVA. LVESV, left ventricular end-systolic volume; LVEDV, left ventricular end-diastolic volume. Color code: for WT RD and WT HFD+L-NAME mice, white (baseline), light gray (1 min), medium gray (5 min), dark gray (10 min) after dobutamine injection; for KO-RD and KO HFD+L-NAME mice, light blue (baseline), medium blue (1 min), darker blue (5 min), dark blue (10 min) after dobutamine injection.

Article Snippet: After transfer to nitrocellulose membranes, blots were rinsed in PBS and blocked in Odyssey blocking buffer (LI-CORbio, Lincoln, NE, USA) for 1 h at room temperature and then incubated with TRPC6 rabbit antibody (1:500, ACC-120, Alomone Labs, Jerusalem, Israel) or TRPC3 rabbit antibody (1:500, #77934, Cell Signaling Technology, Danvers, MA, USA), VDAC rabbit antibody (1:1000, #4661, Cell Signaling), GAPDH rabbit antibody (1:2000, #2118, Cell Signaling), caspase 9 rabbit antibody (1:1000, #9504, Cell Signaling), cleaved caspase 9 rabbit antibody (1:1000, #7237, Cell Signaling), PGC-1α rabbit antibody (1:1000, #2178, Cell Signaling) and BNP rabbit antibody (1:1000, PA5-96084, ThermoFisher, Waltham, MA, USA) at 4 °C overnight.

Techniques: Injection

Journal: International Journal of Molecular Sciences

Article Title: TRPC6 Deficiency Attenuates Mitochondrial and Cardiac Dysfunction in Heart Failure with Preserved Ejection Fraction Induced by High-Fat Diet Plus L-NAME

doi: 10.3390/ijms26199383

Figure Lengend Snippet: Changes in cardiac function at 10 min after dobutamine compared to baseline. Changes in ( A ) heart rate, ( B ) ejection fraction, ( C ) stroke volume, ( D ) cardiac output, ( E ) LVESV, and ( F ) LVEDV in WT and TRPC6 KO mice with RD or HFD+ LNAME. Results are expressed as mean ± SEM, n = 5–6, * p < 0.05 between two groups by post hoc test after one-way ANOVA.

Article Snippet: After transfer to nitrocellulose membranes, blots were rinsed in PBS and blocked in Odyssey blocking buffer (LI-CORbio, Lincoln, NE, USA) for 1 h at room temperature and then incubated with TRPC6 rabbit antibody (1:500, ACC-120, Alomone Labs, Jerusalem, Israel) or TRPC3 rabbit antibody (1:500, #77934, Cell Signaling Technology, Danvers, MA, USA), VDAC rabbit antibody (1:1000, #4661, Cell Signaling), GAPDH rabbit antibody (1:2000, #2118, Cell Signaling), caspase 9 rabbit antibody (1:1000, #9504, Cell Signaling), cleaved caspase 9 rabbit antibody (1:1000, #7237, Cell Signaling), PGC-1α rabbit antibody (1:1000, #2178, Cell Signaling) and BNP rabbit antibody (1:1000, PA5-96084, ThermoFisher, Waltham, MA, USA) at 4 °C overnight.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: TRPC6 Deficiency Attenuates Mitochondrial and Cardiac Dysfunction in Heart Failure with Preserved Ejection Fraction Induced by High-Fat Diet Plus L-NAME

doi: 10.3390/ijms26199383

Figure Lengend Snippet: Treadmill running test in WT and TRPC6 KO mice. ( A ) Running time, ( B ) running distance, and ( C ) vertical work in WT and TRPC6 KO mice with RD or HFD+ L-NAME. Results are expressed as mean ± SEM, n = 5, * p < 0.05 between two groups by post hoc test after one-way ANOVA. Bar colors indicate experimental groups: white = WT RD (regular diet), gray = WT HFD+L-NAME, light blue = KO RD, dark blue = KO HFD+L-NAME.

Article Snippet: After transfer to nitrocellulose membranes, blots were rinsed in PBS and blocked in Odyssey blocking buffer (LI-CORbio, Lincoln, NE, USA) for 1 h at room temperature and then incubated with TRPC6 rabbit antibody (1:500, ACC-120, Alomone Labs, Jerusalem, Israel) or TRPC3 rabbit antibody (1:500, #77934, Cell Signaling Technology, Danvers, MA, USA), VDAC rabbit antibody (1:1000, #4661, Cell Signaling), GAPDH rabbit antibody (1:2000, #2118, Cell Signaling), caspase 9 rabbit antibody (1:1000, #9504, Cell Signaling), cleaved caspase 9 rabbit antibody (1:1000, #7237, Cell Signaling), PGC-1α rabbit antibody (1:1000, #2178, Cell Signaling) and BNP rabbit antibody (1:1000, PA5-96084, ThermoFisher, Waltham, MA, USA) at 4 °C overnight.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: TRPC6 Deficiency Attenuates Mitochondrial and Cardiac Dysfunction in Heart Failure with Preserved Ejection Fraction Induced by High-Fat Diet Plus L-NAME

doi: 10.3390/ijms26199383

Figure Lengend Snippet: Changes in protein expression within the cytosol and mitochondria isolated from the left ventricle of WT and TRPC6 KO mice. ( A ) Representative blots for caspase 9, cleaved caspase 9, PGC1a, TRPC6, TRPC3, BNP, and GAPDH in the cytosol fraction. ( B ) Quantitative analysis results for protein expression levels in the cytosol. ( C ) Representative blots for TRPC6, TRPC3, and VDAC in mitochondria isolation. ( D ) Quantitative analysis results for protein expression levels in mitochondria. Results are expressed as mean ± SEM, n = 3–6, * p < 0.05 between two groups by post hoc test after one-way ANOVA. Bar colors indicate experimental groups: white = WT RD (regular diet), gray = WT HFD+L-NAME, light blue = KO RD, dark blue = KO HFD+L-NAME.

Article Snippet: After transfer to nitrocellulose membranes, blots were rinsed in PBS and blocked in Odyssey blocking buffer (LI-CORbio, Lincoln, NE, USA) for 1 h at room temperature and then incubated with TRPC6 rabbit antibody (1:500, ACC-120, Alomone Labs, Jerusalem, Israel) or TRPC3 rabbit antibody (1:500, #77934, Cell Signaling Technology, Danvers, MA, USA), VDAC rabbit antibody (1:1000, #4661, Cell Signaling), GAPDH rabbit antibody (1:2000, #2118, Cell Signaling), caspase 9 rabbit antibody (1:1000, #9504, Cell Signaling), cleaved caspase 9 rabbit antibody (1:1000, #7237, Cell Signaling), PGC-1α rabbit antibody (1:1000, #2178, Cell Signaling) and BNP rabbit antibody (1:1000, PA5-96084, ThermoFisher, Waltham, MA, USA) at 4 °C overnight.

Techniques: Expressing, Isolation

Journal: International Journal of Molecular Sciences

Article Title: TRPC6 Deficiency Attenuates Mitochondrial and Cardiac Dysfunction in Heart Failure with Preserved Ejection Fraction Induced by High-Fat Diet Plus L-NAME

doi: 10.3390/ijms26199383

Figure Lengend Snippet: Immunohistochemistry staining of BNP and cleaved caspase 9 from the left ventricle of WT and TRPC6 KO mice with RD or HFD+ L-NAME. Representative images for BNP staining in WT RD ( A , E ), WT HFD+ L-NAME ( B , F ), KO RD ( C , G ), and KO HFD+ L-NAME ( D , H ), and cleaved caspase 9 staining in WT RD ( I , M ), WT HFD+ L-NAME ( J , N ), KO RD ( K , O ), and KO HFD+ L-NAME ( L , P ). Upper panel, ×40 magnification; lower panel, boxed regions in ×200 magnification.

Article Snippet: After transfer to nitrocellulose membranes, blots were rinsed in PBS and blocked in Odyssey blocking buffer (LI-CORbio, Lincoln, NE, USA) for 1 h at room temperature and then incubated with TRPC6 rabbit antibody (1:500, ACC-120, Alomone Labs, Jerusalem, Israel) or TRPC3 rabbit antibody (1:500, #77934, Cell Signaling Technology, Danvers, MA, USA), VDAC rabbit antibody (1:1000, #4661, Cell Signaling), GAPDH rabbit antibody (1:2000, #2118, Cell Signaling), caspase 9 rabbit antibody (1:1000, #9504, Cell Signaling), cleaved caspase 9 rabbit antibody (1:1000, #7237, Cell Signaling), PGC-1α rabbit antibody (1:1000, #2178, Cell Signaling) and BNP rabbit antibody (1:1000, PA5-96084, ThermoFisher, Waltham, MA, USA) at 4 °C overnight.

Techniques: Immunohistochemistry, Staining

Journal: International Journal of Molecular Sciences

Article Title: TRPC6 Deficiency Attenuates Mitochondrial and Cardiac Dysfunction in Heart Failure with Preserved Ejection Fraction Induced by High-Fat Diet Plus L-NAME

doi: 10.3390/ijms26199383

Figure Lengend Snippet: Mitochondrial respiration, mitochondria-derived H 2 O 2 production, and mitochondrial DNA copy numbers in WT and TRPC6 KO mice. ( A ) Maximal oxygen consumption rate, ( B ) ATP-linked oxygen consumption rate, ( C ) basal H 2 O 2 generation rate, and ( D ) maximal H 2 O 2 generation rate measurements by Oroboros respirometer in WT and TRPC6 KO mice with RD or HFD+ L-NAME. ( E , F ) Mitochondrial DNA copy number normalized to 18s rRNA and lipoprotein lipase, respectively. Results are expressed as mean ± SEM, n = 6, * p < 0.05 between two groups by post hoc test after one-way ANOVA. Bar colors indicate experimental groups: white = WT RD (regular diet), gray = WT HFD+L-NAME, light blue = KO RD, dark blue = KO HFD+L-NAME.

Article Snippet: After transfer to nitrocellulose membranes, blots were rinsed in PBS and blocked in Odyssey blocking buffer (LI-CORbio, Lincoln, NE, USA) for 1 h at room temperature and then incubated with TRPC6 rabbit antibody (1:500, ACC-120, Alomone Labs, Jerusalem, Israel) or TRPC3 rabbit antibody (1:500, #77934, Cell Signaling Technology, Danvers, MA, USA), VDAC rabbit antibody (1:1000, #4661, Cell Signaling), GAPDH rabbit antibody (1:2000, #2118, Cell Signaling), caspase 9 rabbit antibody (1:1000, #9504, Cell Signaling), cleaved caspase 9 rabbit antibody (1:1000, #7237, Cell Signaling), PGC-1α rabbit antibody (1:1000, #2178, Cell Signaling) and BNP rabbit antibody (1:1000, PA5-96084, ThermoFisher, Waltham, MA, USA) at 4 °C overnight.

Techniques: Derivative Assay