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Figure 4. PKA phosphorylates huntingtin at Ser2550. (A) Bar graph showing MS/MS results of each phosphopeptide from Q23-huntingtin (HTT) purified from Sf9 insect cells incubated without (open bar) or with purified <t>PRKACA</t> (black bar). (B) The multiple sequence alignment compares the human huntingtin amino acid sequence focused on the region of Ser2550 (Red bold) with huntingtins predicted from the gene sequence of other orthologs, Macaca mulatta, Canis lupus familiaris, Mus musculus and Danio rerio. Shading denotes the conservation of PKA substrate motif (RKXS) across these organisms. (C) Immunoblot showing the result of detecting pSer2550 in FLAG-tagged huntingtin overexpressed without or with PRKACA overexpression in HTT null HEK293T cells for 24 h, with abHTT-pS2550, as well as huntingtin level detected with anti-huntingtin reagent (MAB2166), and PRKACA level detected with anti-PRKACA reagent, and α-tubulin as loading control with anti-α-tubulin reagent. (D) Plot showing relative abundance of pSer2550 endogenous huntingtin peptide detected by PRM assay with or without overexpression of PRKACA for 24 h in HEK293T cells. Endogenous huntingtin was enriched by immunoprecipitation with MAB2166 antibody before PRM assay.

Human Prkaca Cdna, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Huntingtin turnover: modulation of huntingtin degradation by cAMP-dependent protein kinase A (PKA) phosphorylation of C-HEAT domain Ser2550."

Article Title: Huntingtin turnover: modulation of huntingtin degradation by cAMP-dependent protein kinase A (PKA) phosphorylation of C-HEAT domain Ser2550.

Journal: Human molecular genetics

doi: 10.1093/hmg/ddac165

Figure Legend Snippet: Figure 4. PKA phosphorylates huntingtin at Ser2550. (A) Bar graph showing MS/MS results of each phosphopeptide from Q23-huntingtin (HTT) purified from Sf9 insect cells incubated without (open bar) or with purified PRKACA (black bar). (B) The multiple sequence alignment compares the human huntingtin amino acid sequence focused on the region of Ser2550 (Red bold) with huntingtins predicted from the gene sequence of other orthologs, Macaca mulatta, Canis lupus familiaris, Mus musculus and Danio rerio. Shading denotes the conservation of PKA substrate motif (RKXS) across these organisms. (C) Immunoblot showing the result of detecting pSer2550 in FLAG-tagged huntingtin overexpressed without or with PRKACA overexpression in HTT null HEK293T cells for 24 h, with abHTT-pS2550, as well as huntingtin level detected with anti-huntingtin reagent (MAB2166), and PRKACA level detected with anti-PRKACA reagent, and α-tubulin as loading control with anti-α-tubulin reagent. (D) Plot showing relative abundance of pSer2550 endogenous huntingtin peptide detected by PRM assay with or without overexpression of PRKACA for 24 h in HEK293T cells. Endogenous huntingtin was enriched by immunoprecipitation with MAB2166 antibody before PRM assay.

Techniques Used: Tandem Mass Spectroscopy, Phospho-proteomics, Purification, Incubation, Sequencing, Western Blot, Over Expression, Control, Immunoprecipitation

Figure 5. PKA Ser2550 phosphorylation increases the rate of huntingtin degradation. (A) The immunoblot (left) shows the results of detecting Q23- huntingtin (HTT) or Q23-huntingtin phosphomutant S2550A overexpressed in HTT null HEK293T cells in the absence or presence of PRKACA for 24 h or 48 h, with anti-huntingtin reagent (MAB2166) and PRKACA level detected with anti-PRKACA reagent, and α-tubulin as loading control with anti-α-tubulin reagent. The histogram (right) shows the huntingtin/α-tubulin band intensities normalized to the mean ratio of each sample at post 24 h transfection. Data represent mean ± s.e.m (n = 2). (B and C) Immunoblots (left) showing cycloheximide (CHX) chase experiments of normal Q23-huntingtin (Q23 HTT) (B) and mutant Q78-huntingtin (Q78 HTT) (C) overexpressed without or with PRKACA overexpression in HTT null HEK293T cells, probed with anti- huntingtin reagent (MAB2166), anti-PRKACA reagent and anti-α-tubulin reagent for huntingtin, PRKACA and α-tubulin as loading control, respectively. For each, the plots (right) show the huntingtin/α-tubulin band intensities normalized to the mean ratio of each sample at post 24-h transfection (P0) in the absence (open symbol) and presence (closed symbol) of PRKACA overexpression. Data represent mean ± s.e.m (n = 2). Asterisks indicate level of statistical significance (paired Student’s t-test, two tailed); ∗P < 0.05.

Figure Legend Snippet: Figure 5. PKA Ser2550 phosphorylation increases the rate of huntingtin degradation. (A) The immunoblot (left) shows the results of detecting Q23- huntingtin (HTT) or Q23-huntingtin phosphomutant S2550A overexpressed in HTT null HEK293T cells in the absence or presence of PRKACA for 24 h or 48 h, with anti-huntingtin reagent (MAB2166) and PRKACA level detected with anti-PRKACA reagent, and α-tubulin as loading control with anti-α-tubulin reagent. The histogram (right) shows the huntingtin/α-tubulin band intensities normalized to the mean ratio of each sample at post 24 h transfection. Data represent mean ± s.e.m (n = 2). (B and C) Immunoblots (left) showing cycloheximide (CHX) chase experiments of normal Q23-huntingtin (Q23 HTT) (B) and mutant Q78-huntingtin (Q78 HTT) (C) overexpressed without or with PRKACA overexpression in HTT null HEK293T cells, probed with anti- huntingtin reagent (MAB2166), anti-PRKACA reagent and anti-α-tubulin reagent for huntingtin, PRKACA and α-tubulin as loading control, respectively. For each, the plots (right) show the huntingtin/α-tubulin band intensities normalized to the mean ratio of each sample at post 24-h transfection (P0) in the absence (open symbol) and presence (closed symbol) of PRKACA overexpression. Data represent mean ± s.e.m (n = 2). Asterisks indicate level of statistical significance (paired Student’s t-test, two tailed); ∗P < 0.05.

Techniques Used: Phospho-proteomics, Western Blot, Control, Transfection, Mutagenesis, Over Expression, Two Tailed Test

Figure 6. Cellular PKA activity is associated with endogenous huntingtin level. (A) The immunoblot (left) shows the expression levels of endogenous huntingtin (HTT) without or with overexpressed PRKACA in HEK293 cells, detected with anti-huntingtin reagent (MAB2166) and PRKACA levels detected with anti-PRKACA reagent and α-tubulin as loading control with anti-α-tubulin reagent. The histogram (right) shows the huntingtin/α-tubulin band intensities normalized to the mean ratio of each sample of empty vector for empty vector (open bar) and PRKACA (closed bar) expressed samples. Data represent mean ± s.e.m (n = 3). (B) The immunoblot (left) shows the expression level of endogenous huntingtin in HEK293T cells stably expressing scramble- or PRKACA-shRNAs, detected with anti-huntingtin reagent (MAB2166) and PRKACA levels detected with anti-PRKACA reagent and α-tubulin as loading control with anti-α-tubulin reagent. The histogram (right) shows the huntingtin/α-tubulin band intensities normalized to the mean ratio of each sample expressing scramble-shRNA for scramble-shRNA (open bar) and PRKACA-shRNA (closed bar) expressed samples. Data represent mean ± s.e.m (n = 3). (C) The immunoblot shows the expression level of endogenous huntingtin in hNPC.10 (Q62/Q20) treated with DMSO or 300 μM of 8-Br-cAMP, detected with anti-huntingtin reagent (MAB2166) and phosphor-CREB with anti-phosphor-CREB (S133) reagent, total CREB with anti-CREB reagent and α-tubulin as loading control with anti-α-tubulin reagent. Of note, Q62-huntingtin (red arrow) separated from Q20-huntingtin (black arrow) was also confirmed by probing with 1F8 antibody [data not shown (61)]. A histogram showing total huntingtin/α-tubulin band intensities normalized to the mean ratio of each sample at 0 h for DMSO (open symbol) and 8-Br-cAMP- (closed symbol) treated samples, respectively. Data represent mean ± s.e.m (n = 2). Asterisks indicate level of statistical significance (paired Student’s t-test, two tailed); ∗P < 0.05.

Figure Legend Snippet: Figure 6. Cellular PKA activity is associated with endogenous huntingtin level. (A) The immunoblot (left) shows the expression levels of endogenous huntingtin (HTT) without or with overexpressed PRKACA in HEK293 cells, detected with anti-huntingtin reagent (MAB2166) and PRKACA levels detected with anti-PRKACA reagent and α-tubulin as loading control with anti-α-tubulin reagent. The histogram (right) shows the huntingtin/α-tubulin band intensities normalized to the mean ratio of each sample of empty vector for empty vector (open bar) and PRKACA (closed bar) expressed samples. Data represent mean ± s.e.m (n = 3). (B) The immunoblot (left) shows the expression level of endogenous huntingtin in HEK293T cells stably expressing scramble- or PRKACA-shRNAs, detected with anti-huntingtin reagent (MAB2166) and PRKACA levels detected with anti-PRKACA reagent and α-tubulin as loading control with anti-α-tubulin reagent. The histogram (right) shows the huntingtin/α-tubulin band intensities normalized to the mean ratio of each sample expressing scramble-shRNA for scramble-shRNA (open bar) and PRKACA-shRNA (closed bar) expressed samples. Data represent mean ± s.e.m (n = 3). (C) The immunoblot shows the expression level of endogenous huntingtin in hNPC.10 (Q62/Q20) treated with DMSO or 300 μM of 8-Br-cAMP, detected with anti-huntingtin reagent (MAB2166) and phosphor-CREB with anti-phosphor-CREB (S133) reagent, total CREB with anti-CREB reagent and α-tubulin as loading control with anti-α-tubulin reagent. Of note, Q62-huntingtin (red arrow) separated from Q20-huntingtin (black arrow) was also confirmed by probing with 1F8 antibody [data not shown (61)]. A histogram showing total huntingtin/α-tubulin band intensities normalized to the mean ratio of each sample at 0 h for DMSO (open symbol) and 8-Br-cAMP- (closed symbol) treated samples, respectively. Data represent mean ± s.e.m (n = 2). Asterisks indicate level of statistical significance (paired Student’s t-test, two tailed); ∗P < 0.05.

Techniques Used: Activity Assay, Western Blot, Expressing, Control, Plasmid Preparation, Stable Transfection, shRNA, Two Tailed Test

Figure 7. Inverse correlation between levels of huntingtin and PRKACA in a panel of HD neural progenitor cells. The scatter plot of the endogenous huntingtin ratio (normalized to pooled reference sample) (X-axis) versus endogenous PRKACA ratio (normalized to pooled reference sample) (Y- axis) measured by quantitative LC–MS/MS using TMT 10-plex across each of the 11 members of an hNPC panel shows an inverse correla- tion between huntingtin and PRKACA levels, as indicated by the linear regression line (black line), that is significant (Multiple R-squared: 0.443, Adjusted R-squared: 0.3812; F-statistic: 7.159 on 1 and 9 DF, P-value: 0.02539.)

Figure Legend Snippet: Figure 7. Inverse correlation between levels of huntingtin and PRKACA in a panel of HD neural progenitor cells. The scatter plot of the endogenous huntingtin ratio (normalized to pooled reference sample) (X-axis) versus endogenous PRKACA ratio (normalized to pooled reference sample) (Y- axis) measured by quantitative LC–MS/MS using TMT 10-plex across each of the 11 members of an hNPC panel shows an inverse correla- tion between huntingtin and PRKACA levels, as indicated by the linear regression line (black line), that is significant (Multiple R-squared: 0.443, Adjusted R-squared: 0.3812; F-statistic: 7.159 on 1 and 9 DF, P-value: 0.02539.)

Techniques Used: Liquid Chromatography with Mass Spectroscopy

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Journal: Human molecular genetics

Article Title: Huntingtin turnover: modulation of huntingtin degradation by cAMP-dependent protein kinase A (PKA) phosphorylation of C-HEAT domain Ser2550.

doi: 10.1093/hmg/ddac165

Figure Lengend Snippet: Figure 4. PKA phosphorylates huntingtin at Ser2550. (A) Bar graph showing MS/MS results of each phosphopeptide from Q23-huntingtin (HTT) purified from Sf9 insect cells incubated without (open bar) or with purified PRKACA (black bar). (B) The multiple sequence alignment compares the human huntingtin amino acid sequence focused on the region of Ser2550 (Red bold) with huntingtins predicted from the gene sequence of other orthologs, Macaca mulatta, Canis lupus familiaris, Mus musculus and Danio rerio. Shading denotes the conservation of PKA substrate motif (RKXS) across these organisms. (C) Immunoblot showing the result of detecting pSer2550 in FLAG-tagged huntingtin overexpressed without or with PRKACA overexpression in HTT null HEK293T cells for 24 h, with abHTT-pS2550, as well as huntingtin level detected with anti-huntingtin reagent (MAB2166), and PRKACA level detected with anti-PRKACA reagent, and α-tubulin as loading control with anti-α-tubulin reagent. (D) Plot showing relative abundance of pSer2550 endogenous huntingtin peptide detected by PRM assay with or without overexpression of PRKACA for 24 h in HEK293T cells. Endogenous huntingtin was enriched by immunoprecipitation with MAB2166 antibody before PRM assay.

Article Snippet: The recombinant human PRKACA cDNA was obtained from Addgene (Plasmid #23495) and cloned into between BamHI and D ow nloaded from https://academ ic.oup.com /hm g/article/32/1/30/6652516 by U niversidade Federal de Santa C atarina user on 07 February 2024 KpnI in pcDNA5 vector (Invitrogen).

Techniques: Tandem Mass Spectroscopy, Phospho-proteomics, Purification, Incubation, Sequencing, Western Blot, Over Expression, Control, Immunoprecipitation

Journal: Human molecular genetics

Article Title: Huntingtin turnover: modulation of huntingtin degradation by cAMP-dependent protein kinase A (PKA) phosphorylation of C-HEAT domain Ser2550.

doi: 10.1093/hmg/ddac165

Figure Lengend Snippet: Figure 5. PKA Ser2550 phosphorylation increases the rate of huntingtin degradation. (A) The immunoblot (left) shows the results of detecting Q23- huntingtin (HTT) or Q23-huntingtin phosphomutant S2550A overexpressed in HTT null HEK293T cells in the absence or presence of PRKACA for 24 h or 48 h, with anti-huntingtin reagent (MAB2166) and PRKACA level detected with anti-PRKACA reagent, and α-tubulin as loading control with anti-α-tubulin reagent. The histogram (right) shows the huntingtin/α-tubulin band intensities normalized to the mean ratio of each sample at post 24 h transfection. Data represent mean ± s.e.m (n = 2). (B and C) Immunoblots (left) showing cycloheximide (CHX) chase experiments of normal Q23-huntingtin (Q23 HTT) (B) and mutant Q78-huntingtin (Q78 HTT) (C) overexpressed without or with PRKACA overexpression in HTT null HEK293T cells, probed with anti- huntingtin reagent (MAB2166), anti-PRKACA reagent and anti-α-tubulin reagent for huntingtin, PRKACA and α-tubulin as loading control, respectively. For each, the plots (right) show the huntingtin/α-tubulin band intensities normalized to the mean ratio of each sample at post 24-h transfection (P0) in the absence (open symbol) and presence (closed symbol) of PRKACA overexpression. Data represent mean ± s.e.m (n = 2). Asterisks indicate level of statistical significance (paired Student’s t-test, two tailed); ∗P < 0.05.

Techniques: Phospho-proteomics, Western Blot, Control, Transfection, Mutagenesis, Over Expression, Two Tailed Test

Journal: Human molecular genetics

Article Title: Huntingtin turnover: modulation of huntingtin degradation by cAMP-dependent protein kinase A (PKA) phosphorylation of C-HEAT domain Ser2550.

doi: 10.1093/hmg/ddac165

Figure Lengend Snippet: Figure 6. Cellular PKA activity is associated with endogenous huntingtin level. (A) The immunoblot (left) shows the expression levels of endogenous huntingtin (HTT) without or with overexpressed PRKACA in HEK293 cells, detected with anti-huntingtin reagent (MAB2166) and PRKACA levels detected with anti-PRKACA reagent and α-tubulin as loading control with anti-α-tubulin reagent. The histogram (right) shows the huntingtin/α-tubulin band intensities normalized to the mean ratio of each sample of empty vector for empty vector (open bar) and PRKACA (closed bar) expressed samples. Data represent mean ± s.e.m (n = 3). (B) The immunoblot (left) shows the expression level of endogenous huntingtin in HEK293T cells stably expressing scramble- or PRKACA-shRNAs, detected with anti-huntingtin reagent (MAB2166) and PRKACA levels detected with anti-PRKACA reagent and α-tubulin as loading control with anti-α-tubulin reagent. The histogram (right) shows the huntingtin/α-tubulin band intensities normalized to the mean ratio of each sample expressing scramble-shRNA for scramble-shRNA (open bar) and PRKACA-shRNA (closed bar) expressed samples. Data represent mean ± s.e.m (n = 3). (C) The immunoblot shows the expression level of endogenous huntingtin in hNPC.10 (Q62/Q20) treated with DMSO or 300 μM of 8-Br-cAMP, detected with anti-huntingtin reagent (MAB2166) and phosphor-CREB with anti-phosphor-CREB (S133) reagent, total CREB with anti-CREB reagent and α-tubulin as loading control with anti-α-tubulin reagent. Of note, Q62-huntingtin (red arrow) separated from Q20-huntingtin (black arrow) was also confirmed by probing with 1F8 antibody [data not shown (61)]. A histogram showing total huntingtin/α-tubulin band intensities normalized to the mean ratio of each sample at 0 h for DMSO (open symbol) and 8-Br-cAMP- (closed symbol) treated samples, respectively. Data represent mean ± s.e.m (n = 2). Asterisks indicate level of statistical significance (paired Student’s t-test, two tailed); ∗P < 0.05.

Techniques: Activity Assay, Western Blot, Expressing, Control, Plasmid Preparation, Stable Transfection, shRNA, Two Tailed Test

Journal: Human molecular genetics

Article Title: Huntingtin turnover: modulation of huntingtin degradation by cAMP-dependent protein kinase A (PKA) phosphorylation of C-HEAT domain Ser2550.

doi: 10.1093/hmg/ddac165

Figure Lengend Snippet: Figure 7. Inverse correlation between levels of huntingtin and PRKACA in a panel of HD neural progenitor cells. The scatter plot of the endogenous huntingtin ratio (normalized to pooled reference sample) (X-axis) versus endogenous PRKACA ratio (normalized to pooled reference sample) (Y- axis) measured by quantitative LC–MS/MS using TMT 10-plex across each of the 11 members of an hNPC panel shows an inverse correla- tion between huntingtin and PRKACA levels, as indicated by the linear regression line (black line), that is significant (Multiple R-squared: 0.443, Adjusted R-squared: 0.3812; F-statistic: 7.159 on 1 and 9 DF, P-value: 0.02539.)

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Functional Characterization of PRKAR1A Mutations Reveals a Unique Molecular Mechanism Causing Acrodysostosis but Multiple Mechanisms Causing Carney Complex *

doi: 10.1074/jbc.M115.656553

Figure Lengend Snippet: TABLE 1

Article Snippet: As described previously ( 11 ), the entire cDNA coding sequences of the human PRKAR1A and the mouse PRKACA genes were obtained by RT-PCR and subcloned in either pCDNA3.1 (Invitrogen) or pRLucN2(h) (Biosignal, PerkinElmer Life Sciences) plasmids for PRKAR1A and pYFP-C3 (Clontech/BD Bioscience) for PRKACA.

Techniques: Mutagenesis

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1) Product Images from "Huntingtin turnover: modulation of huntingtin degradation by cAMP-dependent protein kinase A (PKA) phosphorylation of C-HEAT domain Ser2550."

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