anti pkcε antibody  (Santa Cruz Biotechnology)

Journal: Biophysical Journal

Article Title: Roles for PKC signaling in chromaffin cell exocytosis

doi: 10.1016/j.bpj.2024.12.005

Figure Lengend Snippet: Identification of PKC subtypes in chromaffin cells. ( A ) qPCR analysis of PKC isoform expression in mouse chromaffin cells. The expression levels of eight PKC isoforms (PKCα, PKCβ, PKCγ, PKCδ, PKCε, PKCη, PKCθ, and PKCμ) were measured. PKCα, PKCβ, PKCδ, PKCε, and PKCμ transcripts were detected; PKCβ, PKCδ, PKCε, and PKCμ transcripts were the most abundant. Data are presented as mean ± SD from two independent experiments, total using four different samples. ( B ) Western blot analysis confirms the expression of PKCβ, PKCε, and PKCμ in adrenal medulla. Samples were obtained from three WT mice, with each lane representing an individual mouse. β-Actin was used as a loading control.

Article Snippet: Antibodies used include PKCβ (Proteintech, Rosemont, IL, 12919-1-AP), PKCε (Proteintech, 20877-1-AP), PKD/PKCμ (Cell Signaling Technology, Danvers, MA, 90039T), β-actin (Invitrogen, Waltham, MA, MA1-140), goat anti-rabbit IgG (H+L) secondary, HRP (Thermo Scientific, 31,460), and goat anti-mouse IgG1 secondary, HRP (Thermo Scientific, A10551).

Techniques: Expressing, Western Blot, Control

Journal: Biophysical Journal

Article Title: Roles for PKC signaling in chromaffin cell exocytosis

doi: 10.1016/j.bpj.2024.12.005

Figure Lengend Snippet: PKCβ and PKCε, but not PKCμ, knockdown attenuated PACAP-evoked Ca 2+ transients in chromaffin cells. ( A ) Representative trace of %ΔF/F₀ versus time for PKCβ knockdown and scrambled shRNA-transfected cells loaded with Cal520 and stimulated with 500 nM PACAP. The bottom images illustrate the signal changes at different time points in the same cell as shown in the intensity versus time record. ( B ) Knockdown of PKCβ significantly reduced the maximum amplitude of PACAP-induced Ca 2+ transients. PKCβ knockdown cells exhibited an approximately 70% reduction in the maximum %ΔF/F₀. Data are presented as mean ± SD. ( C ) The total spike area of Ca 2+ transients was also significantly decreased in PKCβ knockdown cells compared with scrambled control cells. Chromaffin cells were loaded with 1 μ M Cal520 for 30 min after transfection with the shRNA plasmid for at least 48 h. Knockdown cells were identified by RFP expression from the shRNA vector. Data are from two independent experiments ( n = 22 scrambled, n = 16 PKCβ KD). ( D ) Representative trace of %ΔF/F₀ versus time for PKCε knockdown and scrambled shRNA-transfected cells stimulated with PACAP. The bottom images show fluorescence signal changes at different time points in the same cell as shown in the trace. ( E ) Knockdown of PKCε using shRNA significantly reduced the maximum amplitude of PACAP-induced Ca 2+ transients. PKCε knockdown cells showed an approximately 70% reduction in the maximum %ΔF/F₀ compared with scrambled shRNA control cells. Data are presented as mean ± SD. ( F ) The total spike area of Ca 2+ transients was also significantly decreased in PKCε knockdown cells compared with scrambled control cells. Chromaffin cells were cotransduced with red GECO and the shRNA plasmid. Knockdown cells were identified by GFP expression from the shRNA vector. Data are from two independent experiments ( n = 18 scrambled, n = 24 PKCε KD). ( G ) Representative trace of %ΔF/F₀ versus time for PKCμ knockdown and scrambled shRNA-transfected cells stimulated with PACAP. The bottom images illustrate fluorescence signal changes at different time points in the same cell as shown in the trace. ( H ) Knockdown of PKCμ using shRNA did not significantly affect the maximum amplitude of PACAP-induced Ca 2+ transients. Data are presented as mean ± SD. ( I ) The total spike area of Ca 2+ signals was not significantly reduced in PKCμ knockdown cells compared with scrambled control cells. Chromaffin cells were loaded with 1 μ M Cal590 for 30 min after transfection with the shRNA plasmid for at least 48 h. Knockdown cells were identified by GFP expression from the shRNA plasmid. Data are from two independent experiments ( n = 29 scrambled, n = 25 PKCμ KD). Statistical significance was determined using a two-tailed unpaired t -test. ∗∗∗∗ p < 0.0001; ns, not significant.

Article Snippet: Antibodies used include PKCβ (Proteintech, Rosemont, IL, 12919-1-AP), PKCε (Proteintech, 20877-1-AP), PKD/PKCμ (Cell Signaling Technology, Danvers, MA, 90039T), β-actin (Invitrogen, Waltham, MA, MA1-140), goat anti-rabbit IgG (H+L) secondary, HRP (Thermo Scientific, 31,460), and goat anti-mouse IgG1 secondary, HRP (Thermo Scientific, A10551).

Techniques: Knockdown, shRNA, Transfection, Control, Plasmid Preparation, Expressing, Fluorescence, Two Tailed Test

Journal: Biophysical Journal

Article Title: Roles for PKC signaling in chromaffin cell exocytosis

doi: 10.1016/j.bpj.2024.12.005

Figure Lengend Snippet: Knockdown of PKCβ and PKCε significantly disrupted PACAP-stimulated exocytosis. ( A ) Representative images of a chromaffin cell with granules loaded with FFN511 (abbreviated FFN) before and after stimulation with PACAP for 45 s. Yellow circles indicate the locations where fusion events occurred. ( B ) Knockdown of PKCβ significantly reduced the number of FFN fusion events per μ m 2 compared with scrambled shRNA control cells ( p = 0.001). Data were collected from two independent experiments ( n = 17 for scrambled, n = 16 for PKCβ KD). ( C ) Knockdown of PKCε significantly reduced the number of FFN fusion events per μ m 2 compared with scrambled shRNA control cells ( p < 0.0001). Data were collected from two independent experiments ( n = 29 for scrambled, n = 27 for PKCε KD). Chromaffin cells were loaded with 20 μ M FFN511 for 30 min after transfection with the shRNA plasmid. Knockdown cells were identified by RFP expression and were stimulated by 500 nM PACAP for 45 s. Data are presented as mean ± SD. Statistical significance was determined using a two-tailed unpaired t -test ( ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001).

Article Snippet: Antibodies used include PKCβ (Proteintech, Rosemont, IL, 12919-1-AP), PKCε (Proteintech, 20877-1-AP), PKD/PKCμ (Cell Signaling Technology, Danvers, MA, 90039T), β-actin (Invitrogen, Waltham, MA, MA1-140), goat anti-rabbit IgG (H+L) secondary, HRP (Thermo Scientific, 31,460), and goat anti-mouse IgG1 secondary, HRP (Thermo Scientific, A10551).

Techniques: Knockdown, shRNA, Control, Transfection, Plasmid Preparation, Expressing, Two Tailed Test