Journal: Biophysical Journal

Article Title: The Rab3 GTPase cycle modulates cardiomyocyte exocytosis and atrial natriuretic peptide release

doi: 10.1016/j.bpj.2025.03.013

Figure Lengend Snippet: Secretion of ANP is G ⍺ q-dependent. ( A ) Representative immunoblotting for intracellular ANP and secreted ANP in culture medium from NRCMs treated with PBS or PE for 24 h with or without FR900359 (G ⍺ qi, 1 μM). Coomassie staining was used as loading control for secreted protein, and Gapdh levels were used as loading control for intracellular protein. n = 4 independent experiments. Quantification of ( B ) the ratio of secreted:intracellular proANP and ( C ) Nppa (ANP) mRNA levels in NRCMs treated with or without PE and/or G ⍺ qi. Histograms depict mean ± standard deviation. ( D ) Representative images of NRCMs transduced with adenoviruses for GFP-Rab3a and treated with PBS or PE for 24 h with or without FR900359 (G ⍺ qi, 1 μM). Cells were immunostained for endogenous ANP (magenta). Nuclei were stained blue with DAPI. Scale bar, 10 μm. Quantification of Manders correlation coefficients for ( E ) the fraction of endogenous ANP colocalized with GFP-Rab3a and ( F ) the fraction of GFP-Rab3a colocalized with endogenous ANP. n = 3 independent experiments with 70–100 cells analyzed for each experiment. Individual data points for Manders coefficients obtained from each image are shown, and the mean value for each independent experiment used for statistical testing is depicted by the larger symbols. ∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.001, two-way ANOVA with Tukey’s multiple-comparisons test.

Article Snippet: Samples were boiled in Laemmli buffer and electrophoresed by standard SDS-PAGE (Bio-Rad) and protein gels stained with SimplyBlue SafeStain (Invitrogen, #LC6060) to assess loading of conditioned medium samples or transferred to PVDF membranes (Millipore, #IPFL00010) for western blotting with the following primary antibodies followed by incubation with IRDye secondary antibodies (1:5000) and imaging and quantification on an Odyssey CLx (LiCor Biosciences) as described previously ( , ): GFP (Novus, NB600-597, 1:1000), Gapdh (Fitzgerald, 10RG109A, 1:50,000), or ANP (Millipore, AB5490,1:500).

Techniques: Western Blot, Staining, Control, Standard Deviation, Transduction

Journal: Biophysical Journal

Article Title: The Rab3 GTPase cycle modulates cardiomyocyte exocytosis and atrial natriuretic peptide release

doi: 10.1016/j.bpj.2025.03.013

Figure Lengend Snippet: Rab3a activity is sufficient to elicit ANP secretion by cardiomyocytes. ( A ) Representative immunoblotting of intracellular and secreted ANP protein in culture medium from NRCMs transduced with adenoviruses for GFP-tagged wild-type Rab3a (Rab3a WT ) or constitutively active GTP-bound Rab3a (Rab3a Q81L ), or βGal control. The protein gel was Coomassie stained as a loading control for secreted protein and Gapdh immunoblotting was used as loading control for intracellular protein. n = 3 independent experiments. Quantification of ( B ) the ratio of secreted:intracellular proANP and ( C ) Nppa (ANP) mRNA levels in NRCMs transduced with adenoviruses for GFP-Rab3a WT , GFP-Rab3a Q81L , or βGal control. Values depicted in histograms are mean ± standard deviation. ( D ) Representative images of NRCMs transduced with adenoviruses for GFP-tagged Rab3a WT or Rab3a Q81L , or βGal control and immunostained for endogenous ANP (magenta). Nuclei were stained blue with DAPI. Scale bar, 10 μm. Quantification of Manders coefficients for ( E ) the fraction of endogenous ANP colocalized with GFP-Rab3a and ( F ) the fraction of GFP-Rab3a colocalized with endogenous ANP. n = 3 independent experiments with 70–100 cells analyzed for each experiment. Individual data points for Manders coefficients obtained from each image are shown, and the mean value for each independent experiment used for statistical testing is depicted by the larger symbols. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.001, one-way ANOVA with Tukey’s multiple-comparisons test.

Article Snippet: Samples were boiled in Laemmli buffer and electrophoresed by standard SDS-PAGE (Bio-Rad) and protein gels stained with SimplyBlue SafeStain (Invitrogen, #LC6060) to assess loading of conditioned medium samples or transferred to PVDF membranes (Millipore, #IPFL00010) for western blotting with the following primary antibodies followed by incubation with IRDye secondary antibodies (1:5000) and imaging and quantification on an Odyssey CLx (LiCor Biosciences) as described previously ( , ): GFP (Novus, NB600-597, 1:1000), Gapdh (Fitzgerald, 10RG109A, 1:50,000), or ANP (Millipore, AB5490,1:500).

Techniques: Activity Assay, Western Blot, Transduction, Control, Staining, Standard Deviation

Journal: Biophysical Journal

Article Title: The Rab3 GTPase cycle modulates cardiomyocyte exocytosis and atrial natriuretic peptide release

doi: 10.1016/j.bpj.2025.03.013

Figure Lengend Snippet: G ⍺ q-dependent activation of Rab3a in cardiomyocytes. ( A ) Rab3a-GTP pull-down assays followed by immunoblotting for input Rab3a protein levels and Rab3a-GTP levels (GST-Rim1 1−200 pull-down) in neonatal rat cardiomyocytes (NRCMs) transduced with adenovirus to express GFP-Rab3a and treated with PBS or phenylephrine (PE) for 24 h with or without FR900359 (G ⍺ qi, 1 μM). The higher-molecular-weight anti-GFP immunoreactive band migrating around 50 kDa was used for quantification of GFP-Rab3a, although the lower-molecular-weight band reliably shows a similar relative pattern. Total input GFP-Rab3a protein was normalized to Gapdh levels and GFP-Rab3a-GTP levels were normalized to total GFP-Rab3a protein levels in the input. Representative immunoblots are shown from n = 4 independent NRCM isolations. Also see . ( B ) Representative images of NRCMs transduced with adenovirus for GFP-Rab3a and treated with PBS or PE for 24 h with or without FR900359 (G ⍺ qi, 1 μM). Cells were immunostained for endogenous GM130 (magenta). Nuclei were stained blue with DAPI. Scale bar, 10 μm. Quantification of Mander’s correlation coefficients for ( C ) the fraction of the Golgi marker GM130 colocalized with GFP-Rab3a and ( D ) the fraction of GFP-Rab3a colocalized with GM130. n = 3 independent experiments with 70–100 cells analyzed for each experiment. Individual data points for Manders coefficients obtained from each image are shown, and the mean value for each independent experiment used for statistical testing is depicted by the larger symbols. ∗ p < 0.05, ∗∗∗ p < 0.001, two-way ANOVA with Tukey’s multiple-comparisons test.

Article Snippet: Samples were boiled in Laemmli buffer and electrophoresed by standard SDS-PAGE (Bio-Rad) and protein gels stained with SimplyBlue SafeStain (Invitrogen, #LC6060) to assess loading of conditioned medium samples or transferred to PVDF membranes (Millipore, #IPFL00010) for western blotting with the following primary antibodies followed by incubation with IRDye secondary antibodies (1:5000) and imaging and quantification on an Odyssey CLx (LiCor Biosciences) as described previously ( , ): GFP (Novus, NB600-597, 1:1000), Gapdh (Fitzgerald, 10RG109A, 1:50,000), or ANP (Millipore, AB5490,1:500).

Techniques: Activation Assay, Western Blot, Transduction, Molecular Weight, Staining, Marker

Journal: Biophysical Journal

Article Title: The Rab3 GTPase cycle modulates cardiomyocyte exocytosis and atrial natriuretic peptide release

doi: 10.1016/j.bpj.2025.03.013

Figure Lengend Snippet: Acute G ⍺ q-dependent activation of Rab3a and ANP secretion. ( A ) Rab3a-GTP pull-down assays followed by immunoblotting for input Rab3a protein levels and Rab3a-GTP levels (GST-Rim1 1−200 pull-down) in neonatal rat cardiomyocytes (NRCMs) transduced with adenovirus to express GFP-Rab3a and treated with PBS or PE for 3 h with or without FR900359 (G ⍺ qi, 1 μM). The higher-molecular-weight anti-GFP immunoreactive band migrating around 50 kDa was used for quantification of GFP-Rab3a, although the lower-molecular-weight band reliably shows a similar relative pattern. Total input GFP-Rab3a protein was normalized to Gapdh levels and GFP-Rab3a-GTP levels were normalized to total GFP-Rab3a protein levels in the input. Representative immunoblots are shown from n = 3 independent NRCM isolations. Also see . ( B ) Representative immunoblotting for intracellular ANP and secreted ANP in culture medium from NRCMs treated with PBS or PE for 3 h with or without FR900359 (G ⍺ qi, 1 μM). Coomassie staining was used as loading control for secreted protein and Gapdh levels were used as loading control for intracellular protein. n = 3 independent NRCM isolations. Quantification of ( C ) the ratio of secreted:intracellular proANP and ( D ) Nppa (ANP) mRNA levels in NRCMs treated with or without PE and/or G ⍺ qi for 3 h. Histograms depict mean ± standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, two-way ANOVA with Tukey's multiple-comparisons test.

Article Snippet: Samples were boiled in Laemmli buffer and electrophoresed by standard SDS-PAGE (Bio-Rad) and protein gels stained with SimplyBlue SafeStain (Invitrogen, #LC6060) to assess loading of conditioned medium samples or transferred to PVDF membranes (Millipore, #IPFL00010) for western blotting with the following primary antibodies followed by incubation with IRDye secondary antibodies (1:5000) and imaging and quantification on an Odyssey CLx (LiCor Biosciences) as described previously ( , ): GFP (Novus, NB600-597, 1:1000), Gapdh (Fitzgerald, 10RG109A, 1:50,000), or ANP (Millipore, AB5490,1:500).

Techniques: Activation Assay, Western Blot, Transduction, Molecular Weight, Staining, Control, Standard Deviation

Journal: Biophysical Journal

Article Title: The Rab3 GTPase cycle modulates cardiomyocyte exocytosis and atrial natriuretic peptide release

doi: 10.1016/j.bpj.2025.03.013

Figure Lengend Snippet: Constitutive Rab3a activity promotes Rab3a protein abundance and localization to the cell periphery. ( A ) Rab3-GTP pull-down assays followed by immunoblotting for input Rab3a protein levels and Rab3a-GTP levels (GST-Rim1 1−200 pull-down) in neonatal rat cardiomyocytes (NRCMs) transduced with adenovirus to express GFP-tagged wild-type Rab3a (Rab3a WT ) or constitutively active GTP-bound Rab3a (Rab3a Q81L ), or β-galactosidase control (βGal). The higher-molecular-weight anti-GFP immunoreactive band migrating around 50 kDa was used for quantification of GFP-Rab3a, although the lower-molecular-weight band reliably shows a similar relative pattern. Total Rab3a levels were normalized to Gapdh and Rab3a-GTP levels were normalized to input Rab3a levels. Representative immunoblots are shown from n = 3 independent NRCM isolations. Also see . ( B ) Rab3a mRNA levels in NRCMs transduced with the indicated adenoviruses. Values represent mean ± standard deviation. ( C ) Representative images of NRCMs transduced with adenoviruses for GFP-Rab3a WT , GFP-Rab3a Q81L , or βGal control and immunostained for endogenous GM130 (magenta). Nuclei were stained blue with DAPI. Scale bar, 10 μm. Quantification of Manders correlation coefficients for ( D ) the fraction of the Golgi marker GM130 colocalized with GFP-Rab3a and ( E ) the fraction of GFP-Rab3a colocalized with GM130. n = 3 independent experiments with 70–100 cells analyzed for each experiment. Individual data points for Manders coefficients obtained from each image are shown, and the mean value for each independent experiment used for statistical testing is depicted by the larger symbols. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, one-way ANOVA with Tukey’s multiple-comparisons test.

Article Snippet: Samples were boiled in Laemmli buffer and electrophoresed by standard SDS-PAGE (Bio-Rad) and protein gels stained with SimplyBlue SafeStain (Invitrogen, #LC6060) to assess loading of conditioned medium samples or transferred to PVDF membranes (Millipore, #IPFL00010) for western blotting with the following primary antibodies followed by incubation with IRDye secondary antibodies (1:5000) and imaging and quantification on an Odyssey CLx (LiCor Biosciences) as described previously ( , ): GFP (Novus, NB600-597, 1:1000), Gapdh (Fitzgerald, 10RG109A, 1:50,000), or ANP (Millipore, AB5490,1:500).

Techniques: Activity Assay, Quantitative Proteomics, Western Blot, Transduction, Control, Molecular Weight, Standard Deviation, Staining, Marker