rabbit anti alpha tubulin Search Results


primary antibody polyclonal rabbit anti α tubulin antiserum  (Rockland Immunochemicals)
93
Rockland Immunochemicals primary antibody polyclonal rabbit anti α tubulin antiserum
Primary Antibody Polyclonal Rabbit Anti α Tubulin Antiserum, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti+alpha+tubulin/AFFINITY+PURF%2E+ANTI-ALPHA-TUBULIN(RABBIT/pmc07845644-300-6-15
Average 93 stars, based on 1 article reviews
primary antibody polyclonal rabbit anti α tubulin antiserum - by Bioz Stars, 2026-09
93/100 stars
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rabbit anti α tubulin  (Rockland Immunochemicals)
93
Rockland Immunochemicals rabbit anti α tubulin
BMP signaling in CCN2/CTGF treated HTM-N cells in vitro . (A, B) Verification of BMP signaling activity in HMT-N cells in vitro . Immunoreactivity of pSmad1/5/8 (green) in HMT-N cells was increased after the treatment with 10 ng/mL BMP-4 (A) and 10 ng/mL BMP-7 (B) . Nuclei were stained with Dapi (blue). n = 3 (C) Western blot analysis of pSmad1/5/8 in the cytoplasmic fraction of HTM-N cells after the treatment with 10 ng/mL BMP-4 or BMP-7 for 1 h. Protein synthesis of pSmad1/5/8 was significantly increased after the treatment with BMP-4 for. (n = 5). GAPDH was used to normalize protein synthesis. Data represented as mean ± SD. Right panel shows a representative Western blot. For statistical analysis unpaired two-tailed t -test was used. (D) Western blot analysis of pSmad1/5/8 in the nuclear fraction of HTM-N cells after the treatment with 10 ng/mL BMP-4 or BMP-7 for 1 h. Protein synthesis of pSmad1/5/8 was significantly increased after 1 h with both treatments (n = 5). LaminB1 was used to normalize protein synthesis. Data represented as mean ± SD. Right panel shows a representative Western blot. For statistical analysis unpaired two-tailed t -test was used. (E) Real-time RT-PCR analysis of Bmp-4 and Bmp-7 after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h in HMT-N cells. mRNA expression of Bmp-4 and Bmp-7 was significantly reduced after the treatment with CCN2/CTGF ( Bmp-4 : control n = 4, 50 ng/mL CCN2/CTGF n = 3, 100 ng/mL CCN2/CTGF n = 3; Bmp-7 : control n = 5, 50 ng/mL CCN2/CTGF n = 4, 100 ng/mL CCN2/CTGF n = 3). mRNA expression was normalized to Gnb2l, and mean value of untreated control cells was set to 1. For statistical analysis the Kruskal–Wallis test was used. (F) Real-time RT-PCR analyses of Smad6 , Smad7 , and Id2 after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h in HMT-N cells. mRNA expression of Smad6 was significantly increased after the treatment with 100 ng/mL CCN2/CTGF (n = 6). mRNA expression of Smad7 was significantly increased after the treatment with 100 ng/mL CCN2/CTGF (control: n = 5, 50 ng/mL CCN2/CTGF: n = 5, 100 ng/mL CCN2/CTGF: n = 4). mRNA expression of Id2 was significantly reduced after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 5, 50 ng/mL CCN2/CTGF: n = 5, 100 ng/mL CCN2/CTGF: n = 4). mRNA expression was normalized to Gnb2l, and mean value of untreated control cells was set to 1. For statistical analysis the Kruskal–Wallis test was used. (G) Western blot analyses of BMP-7 after the treatment with 5 ng/mL, 25 ng/mL, 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h in HMT-N cells. Protein synthesis of BMP-7 was significantly reduced after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 4, 5 ng/mL CCN2/CTGF: n = 3, 25 ng/mL CCN2/CTGF: n = 3, 50 ng/mL CCN2/CTGF: n = 3, 100 ng/mL CCN2/CTGF: n = 3). Mean value of wildtype animals (control) was set to 1. <t>α</t> <t>-Tubulin</t> was used to normalize protein synthesis. For statistical analysis the Kruskal–Wallis test was used. (H) Western blot analysis of pSmad1/5/8 after the treatment with 10 ng/mL BMP-4, 60 ng/mL Noggin and 10 ng/mL BMP-4, and 50 ng/mL CCN2/CTGF and 10 ng/mL BMP-4 in HTM-N cells. pSmad1/5/8 protein synthesis was increased after the treatment with BMP-4 (n = 5), compared to untreated control cells and significantly reduced after the treatment with the combination of Noggin and BMP-4 (n = 5) and the combination of CCN2/CTGF and BMP-4 (n = 5), compared to the treatment with BMP-4 only. Mean value of wildtype animals (control) was set to 1. GAPDH was used to normalize protein synthesis. Data represented as mean ± SD. For statistical analysis the One-way ANOVA test was used. * p ≤ 0.05, ** p < 0.01, *** p < 0.001.
Rabbit Anti α Tubulin, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti+alpha+tubulin/Loading+Control+Antibody+Sampler+Kit/pmc10210157-259-51-54
Average 93 stars, based on 1 article reviews
rabbit anti α tubulin - by Bioz Stars, 2026-09
93/100 stars
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rabbit anti α tubulin (1:5000)  (Bioworld Antibodies)
90
Bioworld Antibodies rabbit anti α tubulin (1:5000)
BMP signaling in CCN2/CTGF treated HTM-N cells in vitro . (A, B) Verification of BMP signaling activity in HMT-N cells in vitro . Immunoreactivity of pSmad1/5/8 (green) in HMT-N cells was increased after the treatment with 10 ng/mL BMP-4 (A) and 10 ng/mL BMP-7 (B) . Nuclei were stained with Dapi (blue). n = 3 (C) Western blot analysis of pSmad1/5/8 in the cytoplasmic fraction of HTM-N cells after the treatment with 10 ng/mL BMP-4 or BMP-7 for 1 h. Protein synthesis of pSmad1/5/8 was significantly increased after the treatment with BMP-4 for. (n = 5). GAPDH was used to normalize protein synthesis. Data represented as mean ± SD. Right panel shows a representative Western blot. For statistical analysis unpaired two-tailed t -test was used. (D) Western blot analysis of pSmad1/5/8 in the nuclear fraction of HTM-N cells after the treatment with 10 ng/mL BMP-4 or BMP-7 for 1 h. Protein synthesis of pSmad1/5/8 was significantly increased after 1 h with both treatments (n = 5). LaminB1 was used to normalize protein synthesis. Data represented as mean ± SD. Right panel shows a representative Western blot. For statistical analysis unpaired two-tailed t -test was used. (E) Real-time RT-PCR analysis of Bmp-4 and Bmp-7 after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h in HMT-N cells. mRNA expression of Bmp-4 and Bmp-7 was significantly reduced after the treatment with CCN2/CTGF ( Bmp-4 : control n = 4, 50 ng/mL CCN2/CTGF n = 3, 100 ng/mL CCN2/CTGF n = 3; Bmp-7 : control n = 5, 50 ng/mL CCN2/CTGF n = 4, 100 ng/mL CCN2/CTGF n = 3). mRNA expression was normalized to Gnb2l, and mean value of untreated control cells was set to 1. For statistical analysis the Kruskal–Wallis test was used. (F) Real-time RT-PCR analyses of Smad6 , Smad7 , and Id2 after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h in HMT-N cells. mRNA expression of Smad6 was significantly increased after the treatment with 100 ng/mL CCN2/CTGF (n = 6). mRNA expression of Smad7 was significantly increased after the treatment with 100 ng/mL CCN2/CTGF (control: n = 5, 50 ng/mL CCN2/CTGF: n = 5, 100 ng/mL CCN2/CTGF: n = 4). mRNA expression of Id2 was significantly reduced after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 5, 50 ng/mL CCN2/CTGF: n = 5, 100 ng/mL CCN2/CTGF: n = 4). mRNA expression was normalized to Gnb2l, and mean value of untreated control cells was set to 1. For statistical analysis the Kruskal–Wallis test was used. (G) Western blot analyses of BMP-7 after the treatment with 5 ng/mL, 25 ng/mL, 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h in HMT-N cells. Protein synthesis of BMP-7 was significantly reduced after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 4, 5 ng/mL CCN2/CTGF: n = 3, 25 ng/mL CCN2/CTGF: n = 3, 50 ng/mL CCN2/CTGF: n = 3, 100 ng/mL CCN2/CTGF: n = 3). Mean value of wildtype animals (control) was set to 1. <t>α</t> <t>-Tubulin</t> was used to normalize protein synthesis. For statistical analysis the Kruskal–Wallis test was used. (H) Western blot analysis of pSmad1/5/8 after the treatment with 10 ng/mL BMP-4, 60 ng/mL Noggin and 10 ng/mL BMP-4, and 50 ng/mL CCN2/CTGF and 10 ng/mL BMP-4 in HTM-N cells. pSmad1/5/8 protein synthesis was increased after the treatment with BMP-4 (n = 5), compared to untreated control cells and significantly reduced after the treatment with the combination of Noggin and BMP-4 (n = 5) and the combination of CCN2/CTGF and BMP-4 (n = 5), compared to the treatment with BMP-4 only. Mean value of wildtype animals (control) was set to 1. GAPDH was used to normalize protein synthesis. Data represented as mean ± SD. For statistical analysis the One-way ANOVA test was used. * p ≤ 0.05, ** p < 0.01, *** p < 0.001.
Rabbit Anti α Tubulin (1:5000), supplied by Bioworld Antibodies, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti+alpha+tubulin/rabbit+anti+%CE%B1+tubulin++1+5000+/pmc07754045-76-54-59
Average 90 stars, based on 1 article reviews
rabbit anti α tubulin (1:5000) - by Bioz Stars, 2026-09
90/100 stars
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anti-cleaved caspase 3 antibody  (BioNordika Oy)
90
BioNordika Oy anti-cleaved caspase 3 antibody
BMP signaling in CCN2/CTGF treated HTM-N cells in vitro . (A, B) Verification of BMP signaling activity in HMT-N cells in vitro . Immunoreactivity of pSmad1/5/8 (green) in HMT-N cells was increased after the treatment with 10 ng/mL BMP-4 (A) and 10 ng/mL BMP-7 (B) . Nuclei were stained with Dapi (blue). n = 3 (C) Western blot analysis of pSmad1/5/8 in the cytoplasmic fraction of HTM-N cells after the treatment with 10 ng/mL BMP-4 or BMP-7 for 1 h. Protein synthesis of pSmad1/5/8 was significantly increased after the treatment with BMP-4 for. (n = 5). GAPDH was used to normalize protein synthesis. Data represented as mean ± SD. Right panel shows a representative Western blot. For statistical analysis unpaired two-tailed t -test was used. (D) Western blot analysis of pSmad1/5/8 in the nuclear fraction of HTM-N cells after the treatment with 10 ng/mL BMP-4 or BMP-7 for 1 h. Protein synthesis of pSmad1/5/8 was significantly increased after 1 h with both treatments (n = 5). LaminB1 was used to normalize protein synthesis. Data represented as mean ± SD. Right panel shows a representative Western blot. For statistical analysis unpaired two-tailed t -test was used. (E) Real-time RT-PCR analysis of Bmp-4 and Bmp-7 after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h in HMT-N cells. mRNA expression of Bmp-4 and Bmp-7 was significantly reduced after the treatment with CCN2/CTGF ( Bmp-4 : control n = 4, 50 ng/mL CCN2/CTGF n = 3, 100 ng/mL CCN2/CTGF n = 3; Bmp-7 : control n = 5, 50 ng/mL CCN2/CTGF n = 4, 100 ng/mL CCN2/CTGF n = 3). mRNA expression was normalized to Gnb2l, and mean value of untreated control cells was set to 1. For statistical analysis the Kruskal–Wallis test was used. (F) Real-time RT-PCR analyses of Smad6 , Smad7 , and Id2 after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h in HMT-N cells. mRNA expression of Smad6 was significantly increased after the treatment with 100 ng/mL CCN2/CTGF (n = 6). mRNA expression of Smad7 was significantly increased after the treatment with 100 ng/mL CCN2/CTGF (control: n = 5, 50 ng/mL CCN2/CTGF: n = 5, 100 ng/mL CCN2/CTGF: n = 4). mRNA expression of Id2 was significantly reduced after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 5, 50 ng/mL CCN2/CTGF: n = 5, 100 ng/mL CCN2/CTGF: n = 4). mRNA expression was normalized to Gnb2l, and mean value of untreated control cells was set to 1. For statistical analysis the Kruskal–Wallis test was used. (G) Western blot analyses of BMP-7 after the treatment with 5 ng/mL, 25 ng/mL, 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h in HMT-N cells. Protein synthesis of BMP-7 was significantly reduced after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 4, 5 ng/mL CCN2/CTGF: n = 3, 25 ng/mL CCN2/CTGF: n = 3, 50 ng/mL CCN2/CTGF: n = 3, 100 ng/mL CCN2/CTGF: n = 3). Mean value of wildtype animals (control) was set to 1. <t>α</t> <t>-Tubulin</t> was used to normalize protein synthesis. For statistical analysis the Kruskal–Wallis test was used. (H) Western blot analysis of pSmad1/5/8 after the treatment with 10 ng/mL BMP-4, 60 ng/mL Noggin and 10 ng/mL BMP-4, and 50 ng/mL CCN2/CTGF and 10 ng/mL BMP-4 in HTM-N cells. pSmad1/5/8 protein synthesis was increased after the treatment with BMP-4 (n = 5), compared to untreated control cells and significantly reduced after the treatment with the combination of Noggin and BMP-4 (n = 5) and the combination of CCN2/CTGF and BMP-4 (n = 5), compared to the treatment with BMP-4 only. Mean value of wildtype animals (control) was set to 1. GAPDH was used to normalize protein synthesis. Data represented as mean ± SD. For statistical analysis the One-way ANOVA test was used. * p ≤ 0.05, ** p < 0.01, *** p < 0.001.
Anti Cleaved Caspase 3 Antibody, supplied by BioNordika Oy, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti+alpha+tubulin/rabbit+anti+acetyl+%CE%B1+tubulin++k40+/pmc12066652-119-19-24
Average 90 stars, based on 1 article reviews
anti-cleaved caspase 3 antibody - by Bioz Stars, 2026-09
90/100 stars
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rabbit monoclonal anti-∆c2 α-tubulin rm447  (RevMAb Inc)
90
RevMAb Inc rabbit monoclonal anti-∆c2 α-tubulin rm447
<t>ΔC2-α-tubulin</t> levels increase in TTLΔ HeLa cells. ( A ) Immunoblotting of whole cell lysates prepared from various tissue culture cell <t>lines.</t> <t>ΔC2-α-tubulin</t> was virtually undetectable. ( B ) Detection of CRISPR/Cas9-induced INDELs in the TTL∆ line (clone 1). Three independent INDELs, all of which resulted in early stop codons (underlined), were identified. ( C ) Immunoblotting of whole cell lysates prepared from the parental HeLa and TTL∆ lines. ( D , E ) Detection of ∆Y- and <t>∆C2-α-tubulin</t> in the TTL∆ line by immunofluorescence ( D ) and immunoblotting in cell lysates ( E ). In merged images in ( D ), ∆Y- or ∆C2-α-tubulin is shown in magenta, total α-tubulin (DM1A staining) in yellow, and DNA (DAPI staining) in cyan. Scale bars in ( D ), 25 µm.
Rabbit Monoclonal Anti ∆C2 α Tubulin Rm447, supplied by RevMAb Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti+alpha+tubulin/rabbit+monoclonal+anti+%E2%88%86c2+%CE%B1+tubulin+rm447/pmc09952995-83-47-52
Average 90 stars, based on 1 article reviews
rabbit monoclonal anti-∆c2 α-tubulin rm447 - by Bioz Stars, 2026-09
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rabbit anti-alpha tubulin antibody  (Abfrontier ltd)
90
Abfrontier ltd rabbit anti-alpha tubulin antibody
<t>ΔC2-α-tubulin</t> levels increase in TTLΔ HeLa cells. ( A ) Immunoblotting of whole cell lysates prepared from various tissue culture cell <t>lines.</t> <t>ΔC2-α-tubulin</t> was virtually undetectable. ( B ) Detection of CRISPR/Cas9-induced INDELs in the TTL∆ line (clone 1). Three independent INDELs, all of which resulted in early stop codons (underlined), were identified. ( C ) Immunoblotting of whole cell lysates prepared from the parental HeLa and TTL∆ lines. ( D , E ) Detection of ∆Y- and <t>∆C2-α-tubulin</t> in the TTL∆ line by immunofluorescence ( D ) and immunoblotting in cell lysates ( E ). In merged images in ( D ), ∆Y- or ∆C2-α-tubulin is shown in magenta, total α-tubulin (DM1A staining) in yellow, and DNA (DAPI staining) in cyan. Scale bars in ( D ), 25 µm.
Rabbit Anti Alpha Tubulin Antibody, supplied by Abfrontier ltd, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti+alpha+tubulin/rabbit+anti+alpha+tubulin+antibody/pmc02680683-487-33-37
Average 90 stars, based on 1 article reviews
rabbit anti-alpha tubulin antibody - by Bioz Stars, 2026-09
90/100 stars
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antibody anti-a-tubulin-hrp (rabbit polyclonal)  (MBL Life science)
90
MBL Life science antibody anti-a-tubulin-hrp (rabbit polyclonal)
<t>ΔC2-α-tubulin</t> levels increase in TTLΔ HeLa cells. ( A ) Immunoblotting of whole cell lysates prepared from various tissue culture cell <t>lines.</t> <t>ΔC2-α-tubulin</t> was virtually undetectable. ( B ) Detection of CRISPR/Cas9-induced INDELs in the TTL∆ line (clone 1). Three independent INDELs, all of which resulted in early stop codons (underlined), were identified. ( C ) Immunoblotting of whole cell lysates prepared from the parental HeLa and TTL∆ lines. ( D , E ) Detection of ∆Y- and <t>∆C2-α-tubulin</t> in the TTL∆ line by immunofluorescence ( D ) and immunoblotting in cell lysates ( E ). In merged images in ( D ), ∆Y- or ∆C2-α-tubulin is shown in magenta, total α-tubulin (DM1A staining) in yellow, and DNA (DAPI staining) in cyan. Scale bars in ( D ), 25 µm.
Antibody Anti A Tubulin Hrp (Rabbit Polyclonal), supplied by MBL Life science, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti+alpha+tubulin/rabbit+anti+alpha+tubulin+antibody/10__7554_slash_elife__59994-230-133-137
Average 90 stars, based on 1 article reviews
antibody anti-a-tubulin-hrp (rabbit polyclonal) - by Bioz Stars, 2026-09
90/100 stars
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rabbit anti-acetylated-α-tubulin  (Enzo Biochem)
90
Enzo Biochem rabbit anti-acetylated-α-tubulin
Dose-dependent alteration of Cx43 expression by pan-HDACI . (A,B) Real-time PCR results of Cx43 mRNA expression levels after overnight inhibition by increasing [TSA] (A) and [VOR] (B) relative to control (untreated) values. Experiments were performed in triplicate and the lowest dose of both pan-HDACI produced an insignificant increase in average Cx43 mRNA levels and a significant reduction in Cx43 mRNA levels at the highest doses. (C,D) Representative Cx43 western blots of ventricular cell lysates from TSA (C) or VOR (D) treated cultures. (E,F) Statistical analysis of the protein densitometry scans from three experiments reveal significant dose-dependent reductions in Cx43 protein content. Cx43 protein levels were normalized to a control sample from each experiment <t>with</t> <t>α-tubulin</t> (αT) expression used as an internal control and <t>acetylated</t> <t>α-tubulin</t> (Ac-αT) as a positive indicator for HDAC inhibition.
Rabbit Anti Acetylated α Tubulin, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti+alpha+tubulin/rabbit+anti+acetylated+%CE%B1+tubulin/pmc03625725-76-19-21
Average 90 stars, based on 1 article reviews
rabbit anti-acetylated-α-tubulin - by Bioz Stars, 2026-09
90/100 stars
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chlamydomonas -specific rabbit anti-α-tubulin antibody  (ICN Pharmaceuticals)
90
ICN Pharmaceuticals chlamydomonas -specific rabbit anti-α-tubulin antibody
Dose-dependent alteration of Cx43 expression by pan-HDACI . (A,B) Real-time PCR results of Cx43 mRNA expression levels after overnight inhibition by increasing [TSA] (A) and [VOR] (B) relative to control (untreated) values. Experiments were performed in triplicate and the lowest dose of both pan-HDACI produced an insignificant increase in average Cx43 mRNA levels and a significant reduction in Cx43 mRNA levels at the highest doses. (C,D) Representative Cx43 western blots of ventricular cell lysates from TSA (C) or VOR (D) treated cultures. (E,F) Statistical analysis of the protein densitometry scans from three experiments reveal significant dose-dependent reductions in Cx43 protein content. Cx43 protein levels were normalized to a control sample from each experiment <t>with</t> <t>α-tubulin</t> (αT) expression used as an internal control and <t>acetylated</t> <t>α-tubulin</t> (Ac-αT) as a positive indicator for HDAC inhibition.
Chlamydomonas Specific Rabbit Anti α Tubulin Antibody, supplied by ICN Pharmaceuticals, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti+alpha+tubulin/chlamydomonas++specific+rabbit+anti+%CE%B1+tubulin+antibody/pmc02174175-93-5-34
Average 90 stars, based on 1 article reviews
chlamydomonas -specific rabbit anti-α-tubulin antibody - by Bioz Stars, 2026-09
90/100 stars
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anti-rabbit alpha-tubulin  (abberior instruments)
90
abberior instruments anti-rabbit alpha-tubulin
Dose-dependent alteration of Cx43 expression by pan-HDACI . (A,B) Real-time PCR results of Cx43 mRNA expression levels after overnight inhibition by increasing [TSA] (A) and [VOR] (B) relative to control (untreated) values. Experiments were performed in triplicate and the lowest dose of both pan-HDACI produced an insignificant increase in average Cx43 mRNA levels and a significant reduction in Cx43 mRNA levels at the highest doses. (C,D) Representative Cx43 western blots of ventricular cell lysates from TSA (C) or VOR (D) treated cultures. (E,F) Statistical analysis of the protein densitometry scans from three experiments reveal significant dose-dependent reductions in Cx43 protein content. Cx43 protein levels were normalized to a control sample from each experiment <t>with</t> <t>α-tubulin</t> (αT) expression used as an internal control and <t>acetylated</t> <t>α-tubulin</t> (Ac-αT) as a positive indicator for HDAC inhibition.
Anti Rabbit Alpha Tubulin, supplied by abberior instruments, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti+alpha+tubulin/anti+rabbit+alpha+tubulin/pmc04803349-45-14-35
Average 90 stars, based on 1 article reviews
anti-rabbit alpha-tubulin - by Bioz Stars, 2026-09
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rabbit anti synapsin  (Boster Bio)
91
Boster Bio rabbit anti synapsin
Dose-dependent alteration of Cx43 expression by pan-HDACI . (A,B) Real-time PCR results of Cx43 mRNA expression levels after overnight inhibition by increasing [TSA] (A) and [VOR] (B) relative to control (untreated) values. Experiments were performed in triplicate and the lowest dose of both pan-HDACI produced an insignificant increase in average Cx43 mRNA levels and a significant reduction in Cx43 mRNA levels at the highest doses. (C,D) Representative Cx43 western blots of ventricular cell lysates from TSA (C) or VOR (D) treated cultures. (E,F) Statistical analysis of the protein densitometry scans from three experiments reveal significant dose-dependent reductions in Cx43 protein content. Cx43 protein levels were normalized to a control sample from each experiment <t>with</t> <t>α-tubulin</t> (αT) expression used as an internal control and <t>acetylated</t> <t>α-tubulin</t> (Ac-αT) as a positive indicator for HDAC inhibition.
Rabbit Anti Synapsin, supplied by Boster Bio, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti+alpha+tubulin/Anti-Synapsin+II+Rabbit+Monoclonal+Antibody/pm34626230-83-9-18
Average 91 stars, based on 1 article reviews
rabbit anti synapsin - by Bioz Stars, 2026-09
91/100 stars
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alpha  (Aviva Systems)
90
Aviva Systems alpha
Dose-dependent alteration of Cx43 expression by pan-HDACI . (A,B) Real-time PCR results of Cx43 mRNA expression levels after overnight inhibition by increasing [TSA] (A) and [VOR] (B) relative to control (untreated) values. Experiments were performed in triplicate and the lowest dose of both pan-HDACI produced an insignificant increase in average Cx43 mRNA levels and a significant reduction in Cx43 mRNA levels at the highest doses. (C,D) Representative Cx43 western blots of ventricular cell lysates from TSA (C) or VOR (D) treated cultures. (E,F) Statistical analysis of the protein densitometry scans from three experiments reveal significant dose-dependent reductions in Cx43 protein content. Cx43 protein levels were normalized to a control sample from each experiment <t>with</t> <t>α-tubulin</t> (αT) expression used as an internal control and <t>acetylated</t> <t>α-tubulin</t> (Ac-αT) as a positive indicator for HDAC inhibition.
Alpha, supplied by Aviva Systems, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti+alpha+tubulin/Anti-alpha-Tubulin+(RABBIT)+Antibody/pm27714890-99-57-65
Average 90 stars, based on 1 article reviews
alpha - by Bioz Stars, 2026-09
90/100 stars
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BMP signaling in CCN2/CTGF treated HTM-N cells in vitro . (A, B) Verification of BMP signaling activity in HMT-N cells in vitro . Immunoreactivity of pSmad1/5/8 (green) in HMT-N cells was increased after the treatment with 10 ng/mL BMP-4 (A) and 10 ng/mL BMP-7 (B) . Nuclei were stained with Dapi (blue). n = 3 (C) Western blot analysis of pSmad1/5/8 in the cytoplasmic fraction of HTM-N cells after the treatment with 10 ng/mL BMP-4 or BMP-7 for 1 h. Protein synthesis of pSmad1/5/8 was significantly increased after the treatment with BMP-4 for. (n = 5). GAPDH was used to normalize protein synthesis. Data represented as mean ± SD. Right panel shows a representative Western blot. For statistical analysis unpaired two-tailed t -test was used. (D) Western blot analysis of pSmad1/5/8 in the nuclear fraction of HTM-N cells after the treatment with 10 ng/mL BMP-4 or BMP-7 for 1 h. Protein synthesis of pSmad1/5/8 was significantly increased after 1 h with both treatments (n = 5). LaminB1 was used to normalize protein synthesis. Data represented as mean ± SD. Right panel shows a representative Western blot. For statistical analysis unpaired two-tailed t -test was used. (E) Real-time RT-PCR analysis of Bmp-4 and Bmp-7 after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h in HMT-N cells. mRNA expression of Bmp-4 and Bmp-7 was significantly reduced after the treatment with CCN2/CTGF ( Bmp-4 : control n = 4, 50 ng/mL CCN2/CTGF n = 3, 100 ng/mL CCN2/CTGF n = 3; Bmp-7 : control n = 5, 50 ng/mL CCN2/CTGF n = 4, 100 ng/mL CCN2/CTGF n = 3). mRNA expression was normalized to Gnb2l, and mean value of untreated control cells was set to 1. For statistical analysis the Kruskal–Wallis test was used. (F) Real-time RT-PCR analyses of Smad6 , Smad7 , and Id2 after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h in HMT-N cells. mRNA expression of Smad6 was significantly increased after the treatment with 100 ng/mL CCN2/CTGF (n = 6). mRNA expression of Smad7 was significantly increased after the treatment with 100 ng/mL CCN2/CTGF (control: n = 5, 50 ng/mL CCN2/CTGF: n = 5, 100 ng/mL CCN2/CTGF: n = 4). mRNA expression of Id2 was significantly reduced after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 5, 50 ng/mL CCN2/CTGF: n = 5, 100 ng/mL CCN2/CTGF: n = 4). mRNA expression was normalized to Gnb2l, and mean value of untreated control cells was set to 1. For statistical analysis the Kruskal–Wallis test was used. (G) Western blot analyses of BMP-7 after the treatment with 5 ng/mL, 25 ng/mL, 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h in HMT-N cells. Protein synthesis of BMP-7 was significantly reduced after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 4, 5 ng/mL CCN2/CTGF: n = 3, 25 ng/mL CCN2/CTGF: n = 3, 50 ng/mL CCN2/CTGF: n = 3, 100 ng/mL CCN2/CTGF: n = 3). Mean value of wildtype animals (control) was set to 1. α -Tubulin was used to normalize protein synthesis. For statistical analysis the Kruskal–Wallis test was used. (H) Western blot analysis of pSmad1/5/8 after the treatment with 10 ng/mL BMP-4, 60 ng/mL Noggin and 10 ng/mL BMP-4, and 50 ng/mL CCN2/CTGF and 10 ng/mL BMP-4 in HTM-N cells. pSmad1/5/8 protein synthesis was increased after the treatment with BMP-4 (n = 5), compared to untreated control cells and significantly reduced after the treatment with the combination of Noggin and BMP-4 (n = 5) and the combination of CCN2/CTGF and BMP-4 (n = 5), compared to the treatment with BMP-4 only. Mean value of wildtype animals (control) was set to 1. GAPDH was used to normalize protein synthesis. Data represented as mean ± SD. For statistical analysis the One-way ANOVA test was used. * p ≤ 0.05, ** p < 0.01, *** p < 0.001.

Journal: Frontiers in Molecular Biosciences

Article Title: CCN2/CTGF tip the balance of growth factors towards TGF-β2 in primary open-angle glaucoma

doi: 10.3389/fmolb.2023.1045411

Figure Lengend Snippet: BMP signaling in CCN2/CTGF treated HTM-N cells in vitro . (A, B) Verification of BMP signaling activity in HMT-N cells in vitro . Immunoreactivity of pSmad1/5/8 (green) in HMT-N cells was increased after the treatment with 10 ng/mL BMP-4 (A) and 10 ng/mL BMP-7 (B) . Nuclei were stained with Dapi (blue). n = 3 (C) Western blot analysis of pSmad1/5/8 in the cytoplasmic fraction of HTM-N cells after the treatment with 10 ng/mL BMP-4 or BMP-7 for 1 h. Protein synthesis of pSmad1/5/8 was significantly increased after the treatment with BMP-4 for. (n = 5). GAPDH was used to normalize protein synthesis. Data represented as mean ± SD. Right panel shows a representative Western blot. For statistical analysis unpaired two-tailed t -test was used. (D) Western blot analysis of pSmad1/5/8 in the nuclear fraction of HTM-N cells after the treatment with 10 ng/mL BMP-4 or BMP-7 for 1 h. Protein synthesis of pSmad1/5/8 was significantly increased after 1 h with both treatments (n = 5). LaminB1 was used to normalize protein synthesis. Data represented as mean ± SD. Right panel shows a representative Western blot. For statistical analysis unpaired two-tailed t -test was used. (E) Real-time RT-PCR analysis of Bmp-4 and Bmp-7 after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h in HMT-N cells. mRNA expression of Bmp-4 and Bmp-7 was significantly reduced after the treatment with CCN2/CTGF ( Bmp-4 : control n = 4, 50 ng/mL CCN2/CTGF n = 3, 100 ng/mL CCN2/CTGF n = 3; Bmp-7 : control n = 5, 50 ng/mL CCN2/CTGF n = 4, 100 ng/mL CCN2/CTGF n = 3). mRNA expression was normalized to Gnb2l, and mean value of untreated control cells was set to 1. For statistical analysis the Kruskal–Wallis test was used. (F) Real-time RT-PCR analyses of Smad6 , Smad7 , and Id2 after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h in HMT-N cells. mRNA expression of Smad6 was significantly increased after the treatment with 100 ng/mL CCN2/CTGF (n = 6). mRNA expression of Smad7 was significantly increased after the treatment with 100 ng/mL CCN2/CTGF (control: n = 5, 50 ng/mL CCN2/CTGF: n = 5, 100 ng/mL CCN2/CTGF: n = 4). mRNA expression of Id2 was significantly reduced after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 5, 50 ng/mL CCN2/CTGF: n = 5, 100 ng/mL CCN2/CTGF: n = 4). mRNA expression was normalized to Gnb2l, and mean value of untreated control cells was set to 1. For statistical analysis the Kruskal–Wallis test was used. (G) Western blot analyses of BMP-7 after the treatment with 5 ng/mL, 25 ng/mL, 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h in HMT-N cells. Protein synthesis of BMP-7 was significantly reduced after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 4, 5 ng/mL CCN2/CTGF: n = 3, 25 ng/mL CCN2/CTGF: n = 3, 50 ng/mL CCN2/CTGF: n = 3, 100 ng/mL CCN2/CTGF: n = 3). Mean value of wildtype animals (control) was set to 1. α -Tubulin was used to normalize protein synthesis. For statistical analysis the Kruskal–Wallis test was used. (H) Western blot analysis of pSmad1/5/8 after the treatment with 10 ng/mL BMP-4, 60 ng/mL Noggin and 10 ng/mL BMP-4, and 50 ng/mL CCN2/CTGF and 10 ng/mL BMP-4 in HTM-N cells. pSmad1/5/8 protein synthesis was increased after the treatment with BMP-4 (n = 5), compared to untreated control cells and significantly reduced after the treatment with the combination of Noggin and BMP-4 (n = 5) and the combination of CCN2/CTGF and BMP-4 (n = 5), compared to the treatment with BMP-4 only. Mean value of wildtype animals (control) was set to 1. GAPDH was used to normalize protein synthesis. Data represented as mean ± SD. For statistical analysis the One-way ANOVA test was used. * p ≤ 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Specific antibodies were used as follows: rabbit anti-pSmad1/5/8 (1:1,000, Cell Signaling Technology, Danvers, MA, United States; RRID:AB_331671), goat anti-BMP7 (1:500, Santa Cruz Biotechnology; RRID:AB_2227926), goat anti-BMP4 (1:500, Santa Cruz Biotechnology; RRID:AB_2243391), rabbit anti-Gremlin (1:200, Santa Cruz Biotechnology; RRID:AB_2279266), rabbit anti-TGF- β1 (1:200, Promega), rabbit anti- TGF- β2 (1:200; Santa Cruz Biotechnology), rabbit anti-α-Tubulin (1:2500, Rockland; RRID:AB_2612816), rabbit anti-pSmad2 (1:200, Cell Signaling Technology; RRID:AB_390732), rabbit anti-pSmad3 (1:200, Cell Signaling Technology; RRID:AB_2193207), rabbit anti-GAPDH-HRP (1:5,000, Cell Signaling Technology; RRID:AB_1642205), rabbit anti-LaminB1 (1:1,000; Cell signaling Technology; Cat# 13435, RRID:AB_2737428), chicken anti-goat (HRP, 1:2000, Santa Cruz Biotechnology), chicken anti-goat (AP, 1:2000, Santa Cruz Biotechnology), chicken anti-rabbit (HRP, 1:2000 Cell Signaling Technology) and chicken anti-rabbit (AP, 1:2000, Santa Cruz Biotechnology). α -Tubulin, GAPDH and total protein staining with Coomassie were used as loading control to normalize the signal intensity of the Western blots.

Techniques: In Vitro, Activity Assay, Staining, Western Blot, Two Tailed Test, Quantitative RT-PCR, Expressing

TGF-β signaling in CCN2/CTGF treated HTM-N cells in vitro and in βB1-CTGF1 mice in vivo . (A) Real-time RT-PCR analyses of Tgf-b1 , Tgf-b2 and Ccn2/Ctgf in HTM-N cells after the treatment with 5 ng/mL, 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h. mRNA expression of Tgf-b1 was significantly increased after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 8, 5 ng/mL CCN2/CTGF: n = 8, 50 ng/mL CCN2/CTGF: n = 6, 100 ng/mL CCN2/CTGF: n = 3). Tgf-b2 mRNA was significantly enhanced after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 5, 5 ng/mL CCN2/CTGF: n = 5, 50 ng/mL CCN2/CTGF: n = 5, 100 ng/mL CCN2/CTGF: n = 5). mRNA expression of Ccn2/Ctgf was significantly increased after the treatment with 5 ng/mL and 50 ng/mL CCN2/CTGF (control: n = 6, 5 ng/mL CCN2/CTGF: n = 6, 50 ng/mL CCN2/CTGF: n = 5, 100 ng/mL CCN2/CTGF: n = 6). mRNA expression was normalized to Gnb2l, and mean value of untreated control cells was set to 1. For statistical analysis the Kruskal–Wallis test was used. (B) Western blot analysis of TGF-β1 and TGF-β2 in HTM-N cells after the treatment with 5 ng/mL, 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h. Protein synthesis of TGF-β1 was significantly increased after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (n = 4). The protein synthesis of TGF-β2 was significantly enhanced after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 6, 5 ng/mL CCN2/CTGF: n = 8, 50 ng/mL CCN2/CTGF: n = 6, 100 ng/mL CCN2/CTGF: n = 5). Right panel shows representative Western Blots for the proteins. Mean value of untreated control cells was set at 1. α -Tubulin was used to normalize protein synthesis. For statistical analysis the Kruskal–Wallis test was used. (C) Real-time RT-PCR analysis of Tgf-b1 and Tgf-b2 in the anterior eye segment of 2-month-old βB1-CTGF1 mice and wildtype littermates. mRNA expression of Tgf-b2 was significantly increased in βB1-CTGF mice, compared to wildtype littermates ( Tgf-b1 : WT: n = 8, TG: n = 6; Tgf-b2 : WT: n = 19, TG: n = 19). For statistical analysis the Mann-Whitney test was used. mRNA expression was normalized to Gnb2l, and mean value of untreated control cells was set to 1. (D) Western blot analysis of TGF-β2 in the anterior eye segment of 2-month-old βB1-CTGF mice and wildtype littermates. Protein synthesis of TGF-β2 was significantly increased in βB1-CTGF1 mice, compared to wildtype littermates (TGF-β2: WT: n = 6, TG: n = 6). Right panel shows a representative Western blot. α -Tubulin was used to normalize protein synthesis. For statistical analysis the Mann-Whitney test was used. (E) Immunohistochemical staining of TGF-β2 (red) in the anterior chamber of 2-month-old βB1-CTGF1 mice and wildtype littermates. Immunoreactivity of TGF-β2 was increased in the TM of βB1-CTGF mice1, in comparison to wildtype mice. Nuclei were stained with Dapi (blue). n = 5 (F) Western blot analysis of the phosphorylation of Smad2 and Smad3. Protein synthesis of pSmad2 and pSmad3 was significantly increased in βB1-CTGF mice, compared to wildtype littermates (pSMAD2: WT: n = 6, TG: n = 6; pSmad3: WT: n = 7, TG: n = 7). Right panel shows representative Western Blots for both proteins. α -Tubulin was used to normalize protein synthesis. For statistical analysis the Mann-Whitney test was used. (G) Immunohistochemical staining of pSmad2 (red) in the anterior chamber angle of 2-month-old βB1-CTGF1 mice and wildtype littermates. Immunoreactivity of pSmad2 is increased in the TM of βB1-CTGF1 mice, in comparison to wildtype mice. Nuclei were stained with Dapi (blue). Data represented as mean ± SD. CB: ciliary body, I: iris, C: cornea, TM: trabecular meshwork. n = 5. * p ≤ 0.05, ** p < 0.01, *** p < 0.001.

Journal: Frontiers in Molecular Biosciences

Article Title: CCN2/CTGF tip the balance of growth factors towards TGF-β2 in primary open-angle glaucoma

doi: 10.3389/fmolb.2023.1045411

Figure Lengend Snippet: TGF-β signaling in CCN2/CTGF treated HTM-N cells in vitro and in βB1-CTGF1 mice in vivo . (A) Real-time RT-PCR analyses of Tgf-b1 , Tgf-b2 and Ccn2/Ctgf in HTM-N cells after the treatment with 5 ng/mL, 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h. mRNA expression of Tgf-b1 was significantly increased after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 8, 5 ng/mL CCN2/CTGF: n = 8, 50 ng/mL CCN2/CTGF: n = 6, 100 ng/mL CCN2/CTGF: n = 3). Tgf-b2 mRNA was significantly enhanced after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 5, 5 ng/mL CCN2/CTGF: n = 5, 50 ng/mL CCN2/CTGF: n = 5, 100 ng/mL CCN2/CTGF: n = 5). mRNA expression of Ccn2/Ctgf was significantly increased after the treatment with 5 ng/mL and 50 ng/mL CCN2/CTGF (control: n = 6, 5 ng/mL CCN2/CTGF: n = 6, 50 ng/mL CCN2/CTGF: n = 5, 100 ng/mL CCN2/CTGF: n = 6). mRNA expression was normalized to Gnb2l, and mean value of untreated control cells was set to 1. For statistical analysis the Kruskal–Wallis test was used. (B) Western blot analysis of TGF-β1 and TGF-β2 in HTM-N cells after the treatment with 5 ng/mL, 50 ng/mL and 100 ng/mL CCN2/CTGF for 24 h. Protein synthesis of TGF-β1 was significantly increased after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (n = 4). The protein synthesis of TGF-β2 was significantly enhanced after the treatment with 50 ng/mL and 100 ng/mL CCN2/CTGF (control: n = 6, 5 ng/mL CCN2/CTGF: n = 8, 50 ng/mL CCN2/CTGF: n = 6, 100 ng/mL CCN2/CTGF: n = 5). Right panel shows representative Western Blots for the proteins. Mean value of untreated control cells was set at 1. α -Tubulin was used to normalize protein synthesis. For statistical analysis the Kruskal–Wallis test was used. (C) Real-time RT-PCR analysis of Tgf-b1 and Tgf-b2 in the anterior eye segment of 2-month-old βB1-CTGF1 mice and wildtype littermates. mRNA expression of Tgf-b2 was significantly increased in βB1-CTGF mice, compared to wildtype littermates ( Tgf-b1 : WT: n = 8, TG: n = 6; Tgf-b2 : WT: n = 19, TG: n = 19). For statistical analysis the Mann-Whitney test was used. mRNA expression was normalized to Gnb2l, and mean value of untreated control cells was set to 1. (D) Western blot analysis of TGF-β2 in the anterior eye segment of 2-month-old βB1-CTGF mice and wildtype littermates. Protein synthesis of TGF-β2 was significantly increased in βB1-CTGF1 mice, compared to wildtype littermates (TGF-β2: WT: n = 6, TG: n = 6). Right panel shows a representative Western blot. α -Tubulin was used to normalize protein synthesis. For statistical analysis the Mann-Whitney test was used. (E) Immunohistochemical staining of TGF-β2 (red) in the anterior chamber of 2-month-old βB1-CTGF1 mice and wildtype littermates. Immunoreactivity of TGF-β2 was increased in the TM of βB1-CTGF mice1, in comparison to wildtype mice. Nuclei were stained with Dapi (blue). n = 5 (F) Western blot analysis of the phosphorylation of Smad2 and Smad3. Protein synthesis of pSmad2 and pSmad3 was significantly increased in βB1-CTGF mice, compared to wildtype littermates (pSMAD2: WT: n = 6, TG: n = 6; pSmad3: WT: n = 7, TG: n = 7). Right panel shows representative Western Blots for both proteins. α -Tubulin was used to normalize protein synthesis. For statistical analysis the Mann-Whitney test was used. (G) Immunohistochemical staining of pSmad2 (red) in the anterior chamber angle of 2-month-old βB1-CTGF1 mice and wildtype littermates. Immunoreactivity of pSmad2 is increased in the TM of βB1-CTGF1 mice, in comparison to wildtype mice. Nuclei were stained with Dapi (blue). Data represented as mean ± SD. CB: ciliary body, I: iris, C: cornea, TM: trabecular meshwork. n = 5. * p ≤ 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Specific antibodies were used as follows: rabbit anti-pSmad1/5/8 (1:1,000, Cell Signaling Technology, Danvers, MA, United States; RRID:AB_331671), goat anti-BMP7 (1:500, Santa Cruz Biotechnology; RRID:AB_2227926), goat anti-BMP4 (1:500, Santa Cruz Biotechnology; RRID:AB_2243391), rabbit anti-Gremlin (1:200, Santa Cruz Biotechnology; RRID:AB_2279266), rabbit anti-TGF- β1 (1:200, Promega), rabbit anti- TGF- β2 (1:200; Santa Cruz Biotechnology), rabbit anti-α-Tubulin (1:2500, Rockland; RRID:AB_2612816), rabbit anti-pSmad2 (1:200, Cell Signaling Technology; RRID:AB_390732), rabbit anti-pSmad3 (1:200, Cell Signaling Technology; RRID:AB_2193207), rabbit anti-GAPDH-HRP (1:5,000, Cell Signaling Technology; RRID:AB_1642205), rabbit anti-LaminB1 (1:1,000; Cell signaling Technology; Cat# 13435, RRID:AB_2737428), chicken anti-goat (HRP, 1:2000, Santa Cruz Biotechnology), chicken anti-goat (AP, 1:2000, Santa Cruz Biotechnology), chicken anti-rabbit (HRP, 1:2000 Cell Signaling Technology) and chicken anti-rabbit (AP, 1:2000, Santa Cruz Biotechnology). α -Tubulin, GAPDH and total protein staining with Coomassie were used as loading control to normalize the signal intensity of the Western blots.

Techniques: In Vitro, In Vivo, Quantitative RT-PCR, Expressing, Western Blot, MANN-WHITNEY, Immunohistochemical staining, Staining

CCN2/CTGF induced TGF-β signaling activation is mediated via the Erk- and RhoA/ROCK signaling pathway. (A) Real-time RT-PCR analysis of Tgf-b1 , Tgf-b2 and Ccn2/Ctgf in HTM-N cells after the inhibition of the Erk-pathway. After treatment with CCN2/CTGF expression of Tgf-b1 , Tgf-b2 , and Ccn2/Ctgf was significantly higher than in DMSO treated control cells. Combined treatment with the Mek1/2 inhibitor and CCN2/CTGF did not lead to changes in expression compared to DMSO treated control cells, but the upregulation was significantly inhibited compared to cells treated with CCN2/CTGF only. Tgf-b2 and Ccn2/Ctgf mRNA expression was significantly reduced after the treatment with the Mek1/2 inhibitor only, compared to DMSO treated control cells (n ≥ 4; # p ≤ .05, ## p < 0.01 to DMSO control; * p ≤ 0.05, ** p < 0.01 to CCN2/CTGF treatment only). (B) Western blot analysis of TGF-β1 in HTMN-cells after the inhibition of the Erk-pathway. After the treatment with CCN2/CTGF protein synthesis of TGF-β1 is significantly increased compared to DMSO treated control cells. Combined treatment with the Mek1/2 inhibitor and CCN2/CTGF did not lead to changes in protein synthesis compared to DMSO treated control cells, but the upregulation was significantly blocked compared to cells treated with CCN2/CTGF only (n ≥ 4, # p ≤ 0.05 to DMSO control; * p ≤ 0.05 to CCN2/CTGF treatment only). Right panel shows a representative Western blot. Mean value of untreated control cells was set at 1. α -Tubulin was used to normalize protein synthesis. (C) Western blot analysis of TGF-β2 in HTMN-cells after the inhibition of the Erk-pathway. After the treatment with CCN2/CTGF protein synthesis of TGF-β2 is significantly increased compared to DMSO treated control cells. Combined treatment with the Mek1/2 inhibitor and CCN2/CTGF did not lead to changes in protein synthesis compared to DMSO treated control cells, but the upregulation was significantly blocked compared to cells treated with CCN2/CTGF only (n = 4, # p ≤ 0.05 to DMSO control; * p ≤ 0.05 to CCN2/CTGF treatment only). Total protein stained with Coomassie was used to normalize protein synthesis. Data represented as mean ± SD. Right panel shows a representative Western blot. (D) Real-time RT-PCR analyses of Tgf-b1 , Tgf-b2 , and Ccn2/Ctgf in HTM-N cells after the inhibition of the RhoA/ROCK signaling pathway. After treatment with CCN2/CTGF expression of Tgf-b1 , Tgf-b2 , and Ccn2/Ctgf was significantly higher than in DMSO treated control cells. Combined treatment with Fasudil and CCN2/CTGF did not lead to changes in expression compared to DMSO treated control cells, but the upregulation was significantly inhibited compared to cells treated with CCN2/CTGF only. Tgf-b2 and Ccn2/Ctgf mRNA expression was significantly reduced after the treatment with the Fasudil only, compared to DMSO treated control cells (n ≥ 5; # p ≤ 0.05, ## p < 0.01 to DMSO control; * p ≤ 0.05, ** p < 0.01 to CCN2/CTGF treatment only). (E) Western blot analysis of TGF-β1 in HTMN-cells after the inhibition of the RhoA/ROCK signaling pathway. After the treatment with CCN2/CTGF protein synthesis of TGF-β1 is significantly increased compared to DMSO treated control cells. Combined treatment with Fasudil and CCN2/CTGF did not lead to changes in protein synthesis compared to DMSO treated control cells, but the upregulation was significantly blocked compared to cells treated with CCN2/CTGF only (n = 3, # p ≤ 0.05 to DMSO control; ** p < 0.01 to CCN2/CTGF treatment only). Right panel shows a representative Western blot. Mean value of untreated control cells was set to1. α -Tubulin was used to normalize protein synthesis. (F) Western blot analysis of TGF-β2 in HTMN-cells after the inhibition of the RhoA/ROCK signaling pathway. After the treatment with CCN2/CTGF protein synthesis of TGF-β2 was significantly increased compared to DMSO treated control cells. Combined treatment with Fasudil and CCN2/CTGF did not lead to changes in protein synthesis compared to DMSO treated control cells, but the upregulation was significantly blocked compared to cells treated with CCN2/CTGF only (n = 7, ## p < 0.01 to DMSO control; * p ≤ 0.05, ** p < 0.01 to CCN2/CTGF treatment only). Total protein stained with Coomassie was used to normalize protein synthesis. Data represented as mean ± SD. Right panel shows a representative Western blot. Dotted line indicates the control group treated with the solvent of the inhibitor for the appropriate experiment. For statistical analysis a one-way ANOVA was performed.

Journal: Frontiers in Molecular Biosciences

Article Title: CCN2/CTGF tip the balance of growth factors towards TGF-β2 in primary open-angle glaucoma

doi: 10.3389/fmolb.2023.1045411

Figure Lengend Snippet: CCN2/CTGF induced TGF-β signaling activation is mediated via the Erk- and RhoA/ROCK signaling pathway. (A) Real-time RT-PCR analysis of Tgf-b1 , Tgf-b2 and Ccn2/Ctgf in HTM-N cells after the inhibition of the Erk-pathway. After treatment with CCN2/CTGF expression of Tgf-b1 , Tgf-b2 , and Ccn2/Ctgf was significantly higher than in DMSO treated control cells. Combined treatment with the Mek1/2 inhibitor and CCN2/CTGF did not lead to changes in expression compared to DMSO treated control cells, but the upregulation was significantly inhibited compared to cells treated with CCN2/CTGF only. Tgf-b2 and Ccn2/Ctgf mRNA expression was significantly reduced after the treatment with the Mek1/2 inhibitor only, compared to DMSO treated control cells (n ≥ 4; # p ≤ .05, ## p < 0.01 to DMSO control; * p ≤ 0.05, ** p < 0.01 to CCN2/CTGF treatment only). (B) Western blot analysis of TGF-β1 in HTMN-cells after the inhibition of the Erk-pathway. After the treatment with CCN2/CTGF protein synthesis of TGF-β1 is significantly increased compared to DMSO treated control cells. Combined treatment with the Mek1/2 inhibitor and CCN2/CTGF did not lead to changes in protein synthesis compared to DMSO treated control cells, but the upregulation was significantly blocked compared to cells treated with CCN2/CTGF only (n ≥ 4, # p ≤ 0.05 to DMSO control; * p ≤ 0.05 to CCN2/CTGF treatment only). Right panel shows a representative Western blot. Mean value of untreated control cells was set at 1. α -Tubulin was used to normalize protein synthesis. (C) Western blot analysis of TGF-β2 in HTMN-cells after the inhibition of the Erk-pathway. After the treatment with CCN2/CTGF protein synthesis of TGF-β2 is significantly increased compared to DMSO treated control cells. Combined treatment with the Mek1/2 inhibitor and CCN2/CTGF did not lead to changes in protein synthesis compared to DMSO treated control cells, but the upregulation was significantly blocked compared to cells treated with CCN2/CTGF only (n = 4, # p ≤ 0.05 to DMSO control; * p ≤ 0.05 to CCN2/CTGF treatment only). Total protein stained with Coomassie was used to normalize protein synthesis. Data represented as mean ± SD. Right panel shows a representative Western blot. (D) Real-time RT-PCR analyses of Tgf-b1 , Tgf-b2 , and Ccn2/Ctgf in HTM-N cells after the inhibition of the RhoA/ROCK signaling pathway. After treatment with CCN2/CTGF expression of Tgf-b1 , Tgf-b2 , and Ccn2/Ctgf was significantly higher than in DMSO treated control cells. Combined treatment with Fasudil and CCN2/CTGF did not lead to changes in expression compared to DMSO treated control cells, but the upregulation was significantly inhibited compared to cells treated with CCN2/CTGF only. Tgf-b2 and Ccn2/Ctgf mRNA expression was significantly reduced after the treatment with the Fasudil only, compared to DMSO treated control cells (n ≥ 5; # p ≤ 0.05, ## p < 0.01 to DMSO control; * p ≤ 0.05, ** p < 0.01 to CCN2/CTGF treatment only). (E) Western blot analysis of TGF-β1 in HTMN-cells after the inhibition of the RhoA/ROCK signaling pathway. After the treatment with CCN2/CTGF protein synthesis of TGF-β1 is significantly increased compared to DMSO treated control cells. Combined treatment with Fasudil and CCN2/CTGF did not lead to changes in protein synthesis compared to DMSO treated control cells, but the upregulation was significantly blocked compared to cells treated with CCN2/CTGF only (n = 3, # p ≤ 0.05 to DMSO control; ** p < 0.01 to CCN2/CTGF treatment only). Right panel shows a representative Western blot. Mean value of untreated control cells was set to1. α -Tubulin was used to normalize protein synthesis. (F) Western blot analysis of TGF-β2 in HTMN-cells after the inhibition of the RhoA/ROCK signaling pathway. After the treatment with CCN2/CTGF protein synthesis of TGF-β2 was significantly increased compared to DMSO treated control cells. Combined treatment with Fasudil and CCN2/CTGF did not lead to changes in protein synthesis compared to DMSO treated control cells, but the upregulation was significantly blocked compared to cells treated with CCN2/CTGF only (n = 7, ## p < 0.01 to DMSO control; * p ≤ 0.05, ** p < 0.01 to CCN2/CTGF treatment only). Total protein stained with Coomassie was used to normalize protein synthesis. Data represented as mean ± SD. Right panel shows a representative Western blot. Dotted line indicates the control group treated with the solvent of the inhibitor for the appropriate experiment. For statistical analysis a one-way ANOVA was performed.

Article Snippet: Specific antibodies were used as follows: rabbit anti-pSmad1/5/8 (1:1,000, Cell Signaling Technology, Danvers, MA, United States; RRID:AB_331671), goat anti-BMP7 (1:500, Santa Cruz Biotechnology; RRID:AB_2227926), goat anti-BMP4 (1:500, Santa Cruz Biotechnology; RRID:AB_2243391), rabbit anti-Gremlin (1:200, Santa Cruz Biotechnology; RRID:AB_2279266), rabbit anti-TGF- β1 (1:200, Promega), rabbit anti- TGF- β2 (1:200; Santa Cruz Biotechnology), rabbit anti-α-Tubulin (1:2500, Rockland; RRID:AB_2612816), rabbit anti-pSmad2 (1:200, Cell Signaling Technology; RRID:AB_390732), rabbit anti-pSmad3 (1:200, Cell Signaling Technology; RRID:AB_2193207), rabbit anti-GAPDH-HRP (1:5,000, Cell Signaling Technology; RRID:AB_1642205), rabbit anti-LaminB1 (1:1,000; Cell signaling Technology; Cat# 13435, RRID:AB_2737428), chicken anti-goat (HRP, 1:2000, Santa Cruz Biotechnology), chicken anti-goat (AP, 1:2000, Santa Cruz Biotechnology), chicken anti-rabbit (HRP, 1:2000 Cell Signaling Technology) and chicken anti-rabbit (AP, 1:2000, Santa Cruz Biotechnology). α -Tubulin, GAPDH and total protein staining with Coomassie were used as loading control to normalize the signal intensity of the Western blots.

Techniques: Activation Assay, Quantitative RT-PCR, Inhibition, Expressing, Western Blot, Staining

ΔC2-α-tubulin levels increase in TTLΔ HeLa cells. ( A ) Immunoblotting of whole cell lysates prepared from various tissue culture cell lines. ΔC2-α-tubulin was virtually undetectable. ( B ) Detection of CRISPR/Cas9-induced INDELs in the TTL∆ line (clone 1). Three independent INDELs, all of which resulted in early stop codons (underlined), were identified. ( C ) Immunoblotting of whole cell lysates prepared from the parental HeLa and TTL∆ lines. ( D , E ) Detection of ∆Y- and ∆C2-α-tubulin in the TTL∆ line by immunofluorescence ( D ) and immunoblotting in cell lysates ( E ). In merged images in ( D ), ∆Y- or ∆C2-α-tubulin is shown in magenta, total α-tubulin (DM1A staining) in yellow, and DNA (DAPI staining) in cyan. Scale bars in ( D ), 25 µm.

Journal: Biomolecules

Article Title: Mechanistic Analysis of CCP1 in Generating ΔC2 α-Tubulin in Mammalian Cells and Photoreceptor Neurons

doi: 10.3390/biom13020357

Figure Lengend Snippet: ΔC2-α-tubulin levels increase in TTLΔ HeLa cells. ( A ) Immunoblotting of whole cell lysates prepared from various tissue culture cell lines. ΔC2-α-tubulin was virtually undetectable. ( B ) Detection of CRISPR/Cas9-induced INDELs in the TTL∆ line (clone 1). Three independent INDELs, all of which resulted in early stop codons (underlined), were identified. ( C ) Immunoblotting of whole cell lysates prepared from the parental HeLa and TTL∆ lines. ( D , E ) Detection of ∆Y- and ∆C2-α-tubulin in the TTL∆ line by immunofluorescence ( D ) and immunoblotting in cell lysates ( E ). In merged images in ( D ), ∆Y- or ∆C2-α-tubulin is shown in magenta, total α-tubulin (DM1A staining) in yellow, and DNA (DAPI staining) in cyan. Scale bars in ( D ), 25 µm.

Article Snippet: Other antibodies used are as follows: GST antibody (Nacalai, San Diego, CA, USA, Cat# 04435-26), 1:1000; rat monoclonal anti-tyrosinated a-tubulin YL1/2 (Accurate Chemical and Scientific, Carle Place, NY, USA, Cat# YSRTMCA77G), 1:3000; rabbit monoclonal anti-detyrosinated α-tubulin RM444 (RevMAb Biosciences, San Francisco, CA, USA, Cat# 31-1335-00), 0.1 µg/mL; rabbit monoclonal anti-∆C2 α-tubulin RM447 (RevMAb Biosciences, Cat# 31-1339-00), 0.5 µg/mL; mouse monoclonal anti-α-tubulin DM1A (Millipore Sigma, Cat# 05-829), 1:3000; mouse monoclonal anti-GAPDH G-9 (Santa Cruz, Dallas, TX, USA, Cat# sc-365062), 1:2000; anti-TTL (Proteintech, Munich, Germany, Cat# 13618-1-AP), 1:1000; anti-CCP1 (Bethyl Laboratories, Montgomery, TX, USA, Rosemont, IL, USA, Cat# A305-295A), 1:1000; anti-GFP (Invitrogen, Cat# A6455) 1:2000; anti-rabbit Alexa Fluor 488 (Thermo Fisher, Waltham, MA, USA, Cat# A-11034), anti-mouse Alexa Fluor 488 (Thermo Fisher, Cat# A-21202), anti-rat Alexa Fluor 680 (Thermo Fisher, Cat# A-21096), 1:10,000; anti-mouse Alexa Fluor 700 (Thermo Fisher, Cat# A-21036), 1:10,000; anti-rabbit Alexa Fluor 700 (Thermo Fisher, Cat# A-21038), 1:10,000; anti-mouse DyLight 800 (Thermo Fisher Cat# SA5-10176), 1:10,000; anti-rabbit IRDye 800CW (LI-COR, Cat# 926-32211), 1:10,000.

Techniques: Western Blot, CRISPR, Immunofluorescence, Staining

Taxol treatment increases the levels of ΔY-α-tubulin but not ΔC2-α-tubulin in cells. ( A ) Detection of ∆Y- and ∆C2-α-tubulin in taxol-treated HeLa, DLD-1, CHL-1, and TTL∆ cells by immunofluorescence ( A ) and immunoblotting in whole cell lysates ( B ). In merged images in ( A ), ∆Y- or ∆C2-α-tubulin is shown in magenta, total α-tubulin (DM1A staining) in yellow, and DNA (DAPI staining) in cyan. Scale bars in ( A ), 25 µm.

Journal: Biomolecules

Article Title: Mechanistic Analysis of CCP1 in Generating ΔC2 α-Tubulin in Mammalian Cells and Photoreceptor Neurons

doi: 10.3390/biom13020357

Figure Lengend Snippet: Taxol treatment increases the levels of ΔY-α-tubulin but not ΔC2-α-tubulin in cells. ( A ) Detection of ∆Y- and ∆C2-α-tubulin in taxol-treated HeLa, DLD-1, CHL-1, and TTL∆ cells by immunofluorescence ( A ) and immunoblotting in whole cell lysates ( B ). In merged images in ( A ), ∆Y- or ∆C2-α-tubulin is shown in magenta, total α-tubulin (DM1A staining) in yellow, and DNA (DAPI staining) in cyan. Scale bars in ( A ), 25 µm.

Article Snippet: Other antibodies used are as follows: GST antibody (Nacalai, San Diego, CA, USA, Cat# 04435-26), 1:1000; rat monoclonal anti-tyrosinated a-tubulin YL1/2 (Accurate Chemical and Scientific, Carle Place, NY, USA, Cat# YSRTMCA77G), 1:3000; rabbit monoclonal anti-detyrosinated α-tubulin RM444 (RevMAb Biosciences, San Francisco, CA, USA, Cat# 31-1335-00), 0.1 µg/mL; rabbit monoclonal anti-∆C2 α-tubulin RM447 (RevMAb Biosciences, Cat# 31-1339-00), 0.5 µg/mL; mouse monoclonal anti-α-tubulin DM1A (Millipore Sigma, Cat# 05-829), 1:3000; mouse monoclonal anti-GAPDH G-9 (Santa Cruz, Dallas, TX, USA, Cat# sc-365062), 1:2000; anti-TTL (Proteintech, Munich, Germany, Cat# 13618-1-AP), 1:1000; anti-CCP1 (Bethyl Laboratories, Montgomery, TX, USA, Rosemont, IL, USA, Cat# A305-295A), 1:1000; anti-GFP (Invitrogen, Cat# A6455) 1:2000; anti-rabbit Alexa Fluor 488 (Thermo Fisher, Waltham, MA, USA, Cat# A-11034), anti-mouse Alexa Fluor 488 (Thermo Fisher, Cat# A-21202), anti-rat Alexa Fluor 680 (Thermo Fisher, Cat# A-21096), 1:10,000; anti-mouse Alexa Fluor 700 (Thermo Fisher, Cat# A-21036), 1:10,000; anti-rabbit Alexa Fluor 700 (Thermo Fisher, Cat# A-21038), 1:10,000; anti-mouse DyLight 800 (Thermo Fisher Cat# SA5-10176), 1:10,000; anti-rabbit IRDye 800CW (LI-COR, Cat# 926-32211), 1:10,000.

Techniques: Immunofluorescence, Western Blot, Staining

CCP1 is the sole peptidase that generates ΔC2-α-tubulin in TTLΔ cells. ( A , B ) siRNA-mediated knock down of CCP1 in TTLΔ cells, followed by detection of ΔC2-α-tubulin with immunofluorescence ( A ) and immunoblotting ( B ). In merged images in ( A ), ∆C2-α-tubulin is shown in magenta, total α-tubulin (DM1A staining) in yellow, and DNA (DAPI staining) in cyan. Scale bars in ( A ), 50 µm. ( C ) Detection of CRISPR/Cas9-induced INDELs in the CCP1 locus in TTL∆ CCP1∆ line. Expected premature stop codons were indicated by underlines. ( D , E ) Detection of ∆C2-α-tubulin in the TTL∆ CCP1∆ line by immunoblotting in cell lysates ( D ) and immunofluorescence ( E ). In merged images in ( E ), ∆Y- or ∆C2-α-tubulin is shown in magenta, total α-tubulin (DM1A staining) in yellow, and DNA (DAPI staining) in cyan. Scale bars in ( E ), 5 µm.

Journal: Biomolecules

Article Title: Mechanistic Analysis of CCP1 in Generating ΔC2 α-Tubulin in Mammalian Cells and Photoreceptor Neurons

doi: 10.3390/biom13020357

Figure Lengend Snippet: CCP1 is the sole peptidase that generates ΔC2-α-tubulin in TTLΔ cells. ( A , B ) siRNA-mediated knock down of CCP1 in TTLΔ cells, followed by detection of ΔC2-α-tubulin with immunofluorescence ( A ) and immunoblotting ( B ). In merged images in ( A ), ∆C2-α-tubulin is shown in magenta, total α-tubulin (DM1A staining) in yellow, and DNA (DAPI staining) in cyan. Scale bars in ( A ), 50 µm. ( C ) Detection of CRISPR/Cas9-induced INDELs in the CCP1 locus in TTL∆ CCP1∆ line. Expected premature stop codons were indicated by underlines. ( D , E ) Detection of ∆C2-α-tubulin in the TTL∆ CCP1∆ line by immunoblotting in cell lysates ( D ) and immunofluorescence ( E ). In merged images in ( E ), ∆Y- or ∆C2-α-tubulin is shown in magenta, total α-tubulin (DM1A staining) in yellow, and DNA (DAPI staining) in cyan. Scale bars in ( E ), 5 µm.

Article Snippet: Other antibodies used are as follows: GST antibody (Nacalai, San Diego, CA, USA, Cat# 04435-26), 1:1000; rat monoclonal anti-tyrosinated a-tubulin YL1/2 (Accurate Chemical and Scientific, Carle Place, NY, USA, Cat# YSRTMCA77G), 1:3000; rabbit monoclonal anti-detyrosinated α-tubulin RM444 (RevMAb Biosciences, San Francisco, CA, USA, Cat# 31-1335-00), 0.1 µg/mL; rabbit monoclonal anti-∆C2 α-tubulin RM447 (RevMAb Biosciences, Cat# 31-1339-00), 0.5 µg/mL; mouse monoclonal anti-α-tubulin DM1A (Millipore Sigma, Cat# 05-829), 1:3000; mouse monoclonal anti-GAPDH G-9 (Santa Cruz, Dallas, TX, USA, Cat# sc-365062), 1:2000; anti-TTL (Proteintech, Munich, Germany, Cat# 13618-1-AP), 1:1000; anti-CCP1 (Bethyl Laboratories, Montgomery, TX, USA, Rosemont, IL, USA, Cat# A305-295A), 1:1000; anti-GFP (Invitrogen, Cat# A6455) 1:2000; anti-rabbit Alexa Fluor 488 (Thermo Fisher, Waltham, MA, USA, Cat# A-11034), anti-mouse Alexa Fluor 488 (Thermo Fisher, Cat# A-21202), anti-rat Alexa Fluor 680 (Thermo Fisher, Cat# A-21096), 1:10,000; anti-mouse Alexa Fluor 700 (Thermo Fisher, Cat# A-21036), 1:10,000; anti-rabbit Alexa Fluor 700 (Thermo Fisher, Cat# A-21038), 1:10,000; anti-mouse DyLight 800 (Thermo Fisher Cat# SA5-10176), 1:10,000; anti-rabbit IRDye 800CW (LI-COR, Cat# 926-32211), 1:10,000.

Techniques: Immunofluorescence, Western Blot, Staining, CRISPR

Overexpression of CCP1 generates ΔC2-α-tubulin more efficiently in cell lines harboring higher levels of ∆Y-α-tubulin. ( A , B ) Detection of ∆C2-α-tubulin in HeLa, HEK293T, or CHL-1 cells transiently expressing EGFP-CCP1 by immunoblotting of cell lysates ( A ) and immunofluorescence ( B ). In merged images in ( B ), total α-tubulin (DM1A staining) is shown in yellow, EGFP in green, ∆C2-α-tubulin in magenta, and DNA (DAPI staining) in blue. Scale bars in ( B ), 20 µm.

Journal: Biomolecules

Article Title: Mechanistic Analysis of CCP1 in Generating ΔC2 α-Tubulin in Mammalian Cells and Photoreceptor Neurons

doi: 10.3390/biom13020357

Figure Lengend Snippet: Overexpression of CCP1 generates ΔC2-α-tubulin more efficiently in cell lines harboring higher levels of ∆Y-α-tubulin. ( A , B ) Detection of ∆C2-α-tubulin in HeLa, HEK293T, or CHL-1 cells transiently expressing EGFP-CCP1 by immunoblotting of cell lysates ( A ) and immunofluorescence ( B ). In merged images in ( B ), total α-tubulin (DM1A staining) is shown in yellow, EGFP in green, ∆C2-α-tubulin in magenta, and DNA (DAPI staining) in blue. Scale bars in ( B ), 20 µm.

Article Snippet: Other antibodies used are as follows: GST antibody (Nacalai, San Diego, CA, USA, Cat# 04435-26), 1:1000; rat monoclonal anti-tyrosinated a-tubulin YL1/2 (Accurate Chemical and Scientific, Carle Place, NY, USA, Cat# YSRTMCA77G), 1:3000; rabbit monoclonal anti-detyrosinated α-tubulin RM444 (RevMAb Biosciences, San Francisco, CA, USA, Cat# 31-1335-00), 0.1 µg/mL; rabbit monoclonal anti-∆C2 α-tubulin RM447 (RevMAb Biosciences, Cat# 31-1339-00), 0.5 µg/mL; mouse monoclonal anti-α-tubulin DM1A (Millipore Sigma, Cat# 05-829), 1:3000; mouse monoclonal anti-GAPDH G-9 (Santa Cruz, Dallas, TX, USA, Cat# sc-365062), 1:2000; anti-TTL (Proteintech, Munich, Germany, Cat# 13618-1-AP), 1:1000; anti-CCP1 (Bethyl Laboratories, Montgomery, TX, USA, Rosemont, IL, USA, Cat# A305-295A), 1:1000; anti-GFP (Invitrogen, Cat# A6455) 1:2000; anti-rabbit Alexa Fluor 488 (Thermo Fisher, Waltham, MA, USA, Cat# A-11034), anti-mouse Alexa Fluor 488 (Thermo Fisher, Cat# A-21202), anti-rat Alexa Fluor 680 (Thermo Fisher, Cat# A-21096), 1:10,000; anti-mouse Alexa Fluor 700 (Thermo Fisher, Cat# A-21036), 1:10,000; anti-rabbit Alexa Fluor 700 (Thermo Fisher, Cat# A-21038), 1:10,000; anti-mouse DyLight 800 (Thermo Fisher Cat# SA5-10176), 1:10,000; anti-rabbit IRDye 800CW (LI-COR, Cat# 926-32211), 1:10,000.

Techniques: Over Expression, Expressing, Western Blot, Immunofluorescence, Staining

CCP1 uses ∆Y-α-tubulin as a substrate to generate ∆C2-α-tubulin in cells. ( A , B ) Detection of ∆C2-α-tubulin in HeLa cells expressing VASH1-SVBP in a doxycycline dependent manner by immunoblotting of cell lysates ( A ) and immunofluorescence ( B ). In merged images in ( B ), total α-tubulin (DM1A staining) is shown in yellow, ∆C2-α-tubulin in magenta, EGFP in green, and DNA (DAPI staining) in blue. Scale bars in ( B ), 20 µm.

Journal: Biomolecules

Article Title: Mechanistic Analysis of CCP1 in Generating ΔC2 α-Tubulin in Mammalian Cells and Photoreceptor Neurons

doi: 10.3390/biom13020357

Figure Lengend Snippet: CCP1 uses ∆Y-α-tubulin as a substrate to generate ∆C2-α-tubulin in cells. ( A , B ) Detection of ∆C2-α-tubulin in HeLa cells expressing VASH1-SVBP in a doxycycline dependent manner by immunoblotting of cell lysates ( A ) and immunofluorescence ( B ). In merged images in ( B ), total α-tubulin (DM1A staining) is shown in yellow, ∆C2-α-tubulin in magenta, EGFP in green, and DNA (DAPI staining) in blue. Scale bars in ( B ), 20 µm.

Article Snippet: Other antibodies used are as follows: GST antibody (Nacalai, San Diego, CA, USA, Cat# 04435-26), 1:1000; rat monoclonal anti-tyrosinated a-tubulin YL1/2 (Accurate Chemical and Scientific, Carle Place, NY, USA, Cat# YSRTMCA77G), 1:3000; rabbit monoclonal anti-detyrosinated α-tubulin RM444 (RevMAb Biosciences, San Francisco, CA, USA, Cat# 31-1335-00), 0.1 µg/mL; rabbit monoclonal anti-∆C2 α-tubulin RM447 (RevMAb Biosciences, Cat# 31-1339-00), 0.5 µg/mL; mouse monoclonal anti-α-tubulin DM1A (Millipore Sigma, Cat# 05-829), 1:3000; mouse monoclonal anti-GAPDH G-9 (Santa Cruz, Dallas, TX, USA, Cat# sc-365062), 1:2000; anti-TTL (Proteintech, Munich, Germany, Cat# 13618-1-AP), 1:1000; anti-CCP1 (Bethyl Laboratories, Montgomery, TX, USA, Rosemont, IL, USA, Cat# A305-295A), 1:1000; anti-GFP (Invitrogen, Cat# A6455) 1:2000; anti-rabbit Alexa Fluor 488 (Thermo Fisher, Waltham, MA, USA, Cat# A-11034), anti-mouse Alexa Fluor 488 (Thermo Fisher, Cat# A-21202), anti-rat Alexa Fluor 680 (Thermo Fisher, Cat# A-21096), 1:10,000; anti-mouse Alexa Fluor 700 (Thermo Fisher, Cat# A-21036), 1:10,000; anti-rabbit Alexa Fluor 700 (Thermo Fisher, Cat# A-21038), 1:10,000; anti-mouse DyLight 800 (Thermo Fisher Cat# SA5-10176), 1:10,000; anti-rabbit IRDye 800CW (LI-COR, Cat# 926-32211), 1:10,000.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining

In ccp1 −/− mouse retinas, ΔC2-α-tubulin staining is reduced distal to the connecting cilium in rod photoreceptor cells. ( A ) Representative images from P15 ccp1 +/+ and ccp1 −/− retinal cross-sections stained with anti-centrin1 (green), WGA (grey), and anti-∆C2-α-tubulin (magenta) antibodies. Scale bars, 5 µm. Abbreviations: outer segment (OS), connecting cilium (CC), and inner segment (IS). ( B ) Representative Airyscan images of isolated outer segments from P15 ccp1 +/+ and ccp1 −/− retinas stained with α-tubulin (cyan), WGA (gray), and anti-∆C2-α-tubulin (magenta) antibodies. Scale bar, 2 µm. Averaged intensity plots shown to right. ( C ) Representative Western blot showing anti-∆C2-α-tubulin and anti-α-tubulin bands from ccp1 +/+ and ccp1 −/− retinal lysates. ∆C2-α-tubulin and α-tubulin levels at P15 were quantified for 3 separate mice and normalized to wild-type ( ccp1 +/+ ) levels. * p = 0.0438; ns, not significant p = 0.8528.

Journal: Biomolecules

Article Title: Mechanistic Analysis of CCP1 in Generating ΔC2 α-Tubulin in Mammalian Cells and Photoreceptor Neurons

doi: 10.3390/biom13020357

Figure Lengend Snippet: In ccp1 −/− mouse retinas, ΔC2-α-tubulin staining is reduced distal to the connecting cilium in rod photoreceptor cells. ( A ) Representative images from P15 ccp1 +/+ and ccp1 −/− retinal cross-sections stained with anti-centrin1 (green), WGA (grey), and anti-∆C2-α-tubulin (magenta) antibodies. Scale bars, 5 µm. Abbreviations: outer segment (OS), connecting cilium (CC), and inner segment (IS). ( B ) Representative Airyscan images of isolated outer segments from P15 ccp1 +/+ and ccp1 −/− retinas stained with α-tubulin (cyan), WGA (gray), and anti-∆C2-α-tubulin (magenta) antibodies. Scale bar, 2 µm. Averaged intensity plots shown to right. ( C ) Representative Western blot showing anti-∆C2-α-tubulin and anti-α-tubulin bands from ccp1 +/+ and ccp1 −/− retinal lysates. ∆C2-α-tubulin and α-tubulin levels at P15 were quantified for 3 separate mice and normalized to wild-type ( ccp1 +/+ ) levels. * p = 0.0438; ns, not significant p = 0.8528.

Article Snippet: Other antibodies used are as follows: GST antibody (Nacalai, San Diego, CA, USA, Cat# 04435-26), 1:1000; rat monoclonal anti-tyrosinated a-tubulin YL1/2 (Accurate Chemical and Scientific, Carle Place, NY, USA, Cat# YSRTMCA77G), 1:3000; rabbit monoclonal anti-detyrosinated α-tubulin RM444 (RevMAb Biosciences, San Francisco, CA, USA, Cat# 31-1335-00), 0.1 µg/mL; rabbit monoclonal anti-∆C2 α-tubulin RM447 (RevMAb Biosciences, Cat# 31-1339-00), 0.5 µg/mL; mouse monoclonal anti-α-tubulin DM1A (Millipore Sigma, Cat# 05-829), 1:3000; mouse monoclonal anti-GAPDH G-9 (Santa Cruz, Dallas, TX, USA, Cat# sc-365062), 1:2000; anti-TTL (Proteintech, Munich, Germany, Cat# 13618-1-AP), 1:1000; anti-CCP1 (Bethyl Laboratories, Montgomery, TX, USA, Rosemont, IL, USA, Cat# A305-295A), 1:1000; anti-GFP (Invitrogen, Cat# A6455) 1:2000; anti-rabbit Alexa Fluor 488 (Thermo Fisher, Waltham, MA, USA, Cat# A-11034), anti-mouse Alexa Fluor 488 (Thermo Fisher, Cat# A-21202), anti-rat Alexa Fluor 680 (Thermo Fisher, Cat# A-21096), 1:10,000; anti-mouse Alexa Fluor 700 (Thermo Fisher, Cat# A-21036), 1:10,000; anti-rabbit Alexa Fluor 700 (Thermo Fisher, Cat# A-21038), 1:10,000; anti-mouse DyLight 800 (Thermo Fisher Cat# SA5-10176), 1:10,000; anti-rabbit IRDye 800CW (LI-COR, Cat# 926-32211), 1:10,000.

Techniques: Staining, Isolation, Western Blot

CCP6 is a candidate for a second peptidase that generates ΔC2-α-tubulin. Immunofluorescence of TTL∆ CCP1∆ cells transiently expressing EGFP-tagged CCPs. In merged images, total α-tubulin (DM1A staining) is shown in yellow, ∆C2-α-tubulin in magenta, EGFP in green, and DNA (DAPI staining) in blue. Scale bars, 10 µm.

Journal: Biomolecules

Article Title: Mechanistic Analysis of CCP1 in Generating ΔC2 α-Tubulin in Mammalian Cells and Photoreceptor Neurons

doi: 10.3390/biom13020357

Figure Lengend Snippet: CCP6 is a candidate for a second peptidase that generates ΔC2-α-tubulin. Immunofluorescence of TTL∆ CCP1∆ cells transiently expressing EGFP-tagged CCPs. In merged images, total α-tubulin (DM1A staining) is shown in yellow, ∆C2-α-tubulin in magenta, EGFP in green, and DNA (DAPI staining) in blue. Scale bars, 10 µm.

Article Snippet: Other antibodies used are as follows: GST antibody (Nacalai, San Diego, CA, USA, Cat# 04435-26), 1:1000; rat monoclonal anti-tyrosinated a-tubulin YL1/2 (Accurate Chemical and Scientific, Carle Place, NY, USA, Cat# YSRTMCA77G), 1:3000; rabbit monoclonal anti-detyrosinated α-tubulin RM444 (RevMAb Biosciences, San Francisco, CA, USA, Cat# 31-1335-00), 0.1 µg/mL; rabbit monoclonal anti-∆C2 α-tubulin RM447 (RevMAb Biosciences, Cat# 31-1339-00), 0.5 µg/mL; mouse monoclonal anti-α-tubulin DM1A (Millipore Sigma, Cat# 05-829), 1:3000; mouse monoclonal anti-GAPDH G-9 (Santa Cruz, Dallas, TX, USA, Cat# sc-365062), 1:2000; anti-TTL (Proteintech, Munich, Germany, Cat# 13618-1-AP), 1:1000; anti-CCP1 (Bethyl Laboratories, Montgomery, TX, USA, Rosemont, IL, USA, Cat# A305-295A), 1:1000; anti-GFP (Invitrogen, Cat# A6455) 1:2000; anti-rabbit Alexa Fluor 488 (Thermo Fisher, Waltham, MA, USA, Cat# A-11034), anti-mouse Alexa Fluor 488 (Thermo Fisher, Cat# A-21202), anti-rat Alexa Fluor 680 (Thermo Fisher, Cat# A-21096), 1:10,000; anti-mouse Alexa Fluor 700 (Thermo Fisher, Cat# A-21036), 1:10,000; anti-rabbit Alexa Fluor 700 (Thermo Fisher, Cat# A-21038), 1:10,000; anti-mouse DyLight 800 (Thermo Fisher Cat# SA5-10176), 1:10,000; anti-rabbit IRDye 800CW (LI-COR, Cat# 926-32211), 1:10,000.

Techniques: Immunofluorescence, Expressing, Staining

Dose-dependent alteration of Cx43 expression by pan-HDACI . (A,B) Real-time PCR results of Cx43 mRNA expression levels after overnight inhibition by increasing [TSA] (A) and [VOR] (B) relative to control (untreated) values. Experiments were performed in triplicate and the lowest dose of both pan-HDACI produced an insignificant increase in average Cx43 mRNA levels and a significant reduction in Cx43 mRNA levels at the highest doses. (C,D) Representative Cx43 western blots of ventricular cell lysates from TSA (C) or VOR (D) treated cultures. (E,F) Statistical analysis of the protein densitometry scans from three experiments reveal significant dose-dependent reductions in Cx43 protein content. Cx43 protein levels were normalized to a control sample from each experiment with α-tubulin (αT) expression used as an internal control and acetylated α-tubulin (Ac-αT) as a positive indicator for HDAC inhibition.

Journal: Frontiers in Pharmacology

Article Title: Histone deacetylase inhibition reduces cardiac connexin43 expression and gap junction communication

doi: 10.3389/fphar.2013.00044

Figure Lengend Snippet: Dose-dependent alteration of Cx43 expression by pan-HDACI . (A,B) Real-time PCR results of Cx43 mRNA expression levels after overnight inhibition by increasing [TSA] (A) and [VOR] (B) relative to control (untreated) values. Experiments were performed in triplicate and the lowest dose of both pan-HDACI produced an insignificant increase in average Cx43 mRNA levels and a significant reduction in Cx43 mRNA levels at the highest doses. (C,D) Representative Cx43 western blots of ventricular cell lysates from TSA (C) or VOR (D) treated cultures. (E,F) Statistical analysis of the protein densitometry scans from three experiments reveal significant dose-dependent reductions in Cx43 protein content. Cx43 protein levels were normalized to a control sample from each experiment with α-tubulin (αT) expression used as an internal control and acetylated α-tubulin (Ac-αT) as a positive indicator for HDAC inhibition.

Article Snippet: Primary antibodies used in this study include rabbit anti-Cx43 (Chemicon), mouse anti-Cx43 (Zymed), mouse anti-Cx40 (Zymed), mouse anti-α-tubulin (Sigma), rabbit anti-acetylated-α-tubulin (Enzo), mouse anti-α-actin antibody (Sigma), rabbit anti-acetylated lysine antibody (Abcam), and Ncad (Sigma). pSer-specific Cx43 antibodies were produced as previously described including rabbit anti-pS255 (Santa Cruz; ), rabbit anti-pS262 (Santa Cruz; ), rabbit anti-pS279/282 (Santa Cruz; ; ), rabbit anti-pS325/328/330 , rabbit anti-pS365 , rabbit anti-pS368 (R&D; ), and rabbit anti-pS373 (P. D. Lampe, personal communication, manuscript in preparation).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Inhibition, Produced, Western Blot

Reduction of Cx40 and N-cadherin expression by HDACI . (A) Overnight (18 h) treatment with 1 μM VOR significantly reduced Cx43 and N-cadherin mRNA levels by 45 ± 5 and 30 ± 6%, respectively. Experimental mean values were statistically different from control values ( p -value < 0.05, one-way ANOVA). (B) Immunoblots were also performed for Ncad and β-actin (internal control). Total Ncad levels from three experiments were decreased by 1 μM VOR relative to control (untreated) samples. (C) Representative Cx40 western blots of atrial cell lysates from control or 1 μM VOR-treated cultures. (D) Statistical analysis of the protein densitometry scans from four experiments reveals a significant reduction in Cx40 protein content. Cx40 protein levels were normalized to a control sample from each experiment with α-tubulin expression used as an internal control and acetylated α-tubulin (Ac-αT) as a positive indicator for HDAC inhibition.

Journal: Frontiers in Pharmacology

Article Title: Histone deacetylase inhibition reduces cardiac connexin43 expression and gap junction communication

doi: 10.3389/fphar.2013.00044

Figure Lengend Snippet: Reduction of Cx40 and N-cadherin expression by HDACI . (A) Overnight (18 h) treatment with 1 μM VOR significantly reduced Cx43 and N-cadherin mRNA levels by 45 ± 5 and 30 ± 6%, respectively. Experimental mean values were statistically different from control values ( p -value < 0.05, one-way ANOVA). (B) Immunoblots were also performed for Ncad and β-actin (internal control). Total Ncad levels from three experiments were decreased by 1 μM VOR relative to control (untreated) samples. (C) Representative Cx40 western blots of atrial cell lysates from control or 1 μM VOR-treated cultures. (D) Statistical analysis of the protein densitometry scans from four experiments reveals a significant reduction in Cx40 protein content. Cx40 protein levels were normalized to a control sample from each experiment with α-tubulin expression used as an internal control and acetylated α-tubulin (Ac-αT) as a positive indicator for HDAC inhibition.

Article Snippet: Primary antibodies used in this study include rabbit anti-Cx43 (Chemicon), mouse anti-Cx43 (Zymed), mouse anti-Cx40 (Zymed), mouse anti-α-tubulin (Sigma), rabbit anti-acetylated-α-tubulin (Enzo), mouse anti-α-actin antibody (Sigma), rabbit anti-acetylated lysine antibody (Abcam), and Ncad (Sigma). pSer-specific Cx43 antibodies were produced as previously described including rabbit anti-pS255 (Santa Cruz; ), rabbit anti-pS262 (Santa Cruz; ), rabbit anti-pS279/282 (Santa Cruz; ; ), rabbit anti-pS325/328/330 , rabbit anti-pS365 , rabbit anti-pS368 (R&D; ), and rabbit anti-pS373 (P. D. Lampe, personal communication, manuscript in preparation).

Techniques: Expressing, Western Blot, Inhibition