primary antibodies for phospho-p42/p44mapk  (Cell Signaling Technology Inc)

Journal: American Journal of Physiology - Renal Physiology

Article Title: Angiotensin II stimulates fibronectin protein synthesis via a Gβγ/arachidonic acid-dependent pathway

doi: 10.1152/ajprenal.00094.2014

Figure Lengend Snippet: Gβγ mediates ANG II-induced c-Src, ERK, and EGFR receptor (EGFR) activity. β-Adrenergic receptor kinase (βARK-ct) or empty vector (mock) plasmids were transfected into rabbit proximal tubule cells 24 h before assay. Proximal tubule cells were treated with 0.1% DMSO (vol/vol), arachidonic acid (AA; 15 μmol/l), eicosatetraenoic acid (ETYA; 15 μmol/l), or ANG II (1 μmol/l) for 5 min or with plasmids encoding β1 and γ2, (5 μg each) for 24 h. Extracts from cells were analyzed by Western blotting using anti-phospho-c-Src (Tyr416), anti-phospho-p42MAPK (Try202)/p44MAPK (Try204) (ERK1/2), p42MAPK (Try202)/p44MAPK (Try204), and anti-EGFR antibodies. Equal protein loading was confirmed by membrane reprobing with anti-c-Src, anti-ERK, and anti-EGFR antibodies. The corresponding bands were scanned, and intensities were normalized to anti-c-Src, anti-ERK, and anti-EGFR from the same tubular cell protein extract. One representative Western blot is shown for every experiment. A: phosphorylation of c-Src (p-c-Src; top) compared with total expression of c-Src (bottom). B: phosphorylation of ERK (p-ERK; top) compared with total expression of ERK (bottom). C: phosphorylation of EGFR (top) compared with total expression of EGFR (bottom). Bar graphs depict the quantitative densitometry analysis for Western blot densitometry data. Values are means ± SE of 4 independent experiments. ns, Not significant compared with control. **P < 0.01 for comparison with control.

Article Snippet: Antibodies against phospho-c-Src (Tyr 416 ), phospho-cPLA 2 (Ser 505 ), phospho-p42MAPK (Try 202 )/p44MAPK (Try 204 ; ERK1/2), p42MAPK (Try 202 )/p44MAPK (Try 204 ), cPLA 2 , and c-Src were purchased from Cell Signaling Technology.

Techniques: Activity Assay, Plasmid Preparation, Transfection, Western Blot, Expressing

Journal: American Journal of Physiology - Renal Physiology

Article Title: Angiotensin II stimulates fibronectin protein synthesis via a Gβγ/arachidonic acid-dependent pathway

doi: 10.1152/ajprenal.00094.2014

Figure Lengend Snippet: Effect of different Gβγ combinations on EGFR, c-Src, and ERK activity. Rabbit proximal tubule cells were transiently transfected without (mock) or with 10 μg of the expression plasmids for Gβ1, Gβ2, Gβ3, Gβ4, or Gγ2. Equal amounts of cell lysates (30 μg) were resolved by SDS-PAGE followed by Western blotting with anti-EGFR, anti-phospho-c-Src (Tyr416), and anti-phospho-p42MAPK (Try202)/p44MAPK (Try204) (ERK1/2), and p42MAPK (Try202)/p44MAPK (Try204) antibodies. Equal protein loading was confirmed by membrane reprobing with anti-EGFR, anti-c-Src, or anti-ERK antibodies. The corresponding bands were scanned, and intensities were normalized to anti-EGFR, anti-c-Src, and anti-ERK from the same tubular cell protein extract. One representative Western blot is shown for every experiment. A: phosphorylation of EGFR (top) compared with total expression of EGFR (bottom). B: phosphorylation of c-Src (p-c-Src; top) compared with total expression of c-Src (bottom). C: phosphorylation of ERK (p-ERK; top) compared with total expression of ERK (bottom). Bar graphs depict the quantitative densitometry analysis for Western blot densitometry data. Values are means ± SE of 4 independent experiments. ***P < 0.001, **P < 0.01 for comparison with control.

Article Snippet: Antibodies against phospho-c-Src (Tyr 416 ), phospho-cPLA 2 (Ser 505 ), phospho-p42MAPK (Try 202 )/p44MAPK (Try 204 ; ERK1/2), p42MAPK (Try 202 )/p44MAPK (Try 204 ), cPLA 2 , and c-Src were purchased from Cell Signaling Technology.

Techniques: Activity Assay, Transfection, Expressing, SDS Page, Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: Angiotensin II stimulates fibronectin protein synthesis via a Gβγ/arachidonic acid-dependent pathway

doi: 10.1152/ajprenal.00094.2014

Figure Lengend Snippet: Gβγ mediates ANG II-induced c-Src, ERK, and EGFR receptor (EGFR) activity. β-Adrenergic receptor kinase (βARK-ct) or empty vector (mock) plasmids were transfected into rabbit proximal tubule cells 24 h before assay. Proximal tubule cells were treated with 0.1% DMSO (vol/vol), arachidonic acid (AA; 15 μmol/l), eicosatetraenoic acid (ETYA; 15 μmol/l), or ANG II (1 μmol/l) for 5 min or with plasmids encoding β1 and γ2, (5 μg each) for 24 h. Extracts from cells were analyzed by Western blotting using anti-phospho-c-Src (Tyr416), anti-phospho-p42MAPK (Try202)/p44MAPK (Try204) (ERK1/2), p42MAPK (Try202)/p44MAPK (Try204), and anti-EGFR antibodies. Equal protein loading was confirmed by membrane reprobing with anti-c-Src, anti-ERK, and anti-EGFR antibodies. The corresponding bands were scanned, and intensities were normalized to anti-c-Src, anti-ERK, and anti-EGFR from the same tubular cell protein extract. One representative Western blot is shown for every experiment. A: phosphorylation of c-Src (p-c-Src; top) compared with total expression of c-Src (bottom). B: phosphorylation of ERK (p-ERK; top) compared with total expression of ERK (bottom). C: phosphorylation of EGFR (top) compared with total expression of EGFR (bottom). Bar graphs depict the quantitative densitometry analysis for Western blot densitometry data. Values are means ± SE of 4 independent experiments. ns, Not significant compared with control. **P < 0.01 for comparison with control.

Article Snippet: Antibodies against phospho-c-Src (Tyr 416 ), phospho-cPLA 2 (Ser 505 ), phospho-p42MAPK (Try 202 )/p44MAPK (Try 204 ; ERK1/2), p42MAPK (Try 202 )/p44MAPK (Try 204 ), cPLA 2 , and c-Src were purchased from Cell Signaling Technology.

Techniques: Activity Assay, Plasmid Preparation, Transfection, Western Blot, Expressing

Journal: American Journal of Physiology - Renal Physiology

Article Title: Angiotensin II stimulates fibronectin protein synthesis via a Gβγ/arachidonic acid-dependent pathway

doi: 10.1152/ajprenal.00094.2014

Figure Lengend Snippet: Effect of different Gβγ combinations on EGFR, c-Src, and ERK activity. Rabbit proximal tubule cells were transiently transfected without (mock) or with 10 μg of the expression plasmids for Gβ1, Gβ2, Gβ3, Gβ4, or Gγ2. Equal amounts of cell lysates (30 μg) were resolved by SDS-PAGE followed by Western blotting with anti-EGFR, anti-phospho-c-Src (Tyr416), and anti-phospho-p42MAPK (Try202)/p44MAPK (Try204) (ERK1/2), and p42MAPK (Try202)/p44MAPK (Try204) antibodies. Equal protein loading was confirmed by membrane reprobing with anti-EGFR, anti-c-Src, or anti-ERK antibodies. The corresponding bands were scanned, and intensities were normalized to anti-EGFR, anti-c-Src, and anti-ERK from the same tubular cell protein extract. One representative Western blot is shown for every experiment. A: phosphorylation of EGFR (top) compared with total expression of EGFR (bottom). B: phosphorylation of c-Src (p-c-Src; top) compared with total expression of c-Src (bottom). C: phosphorylation of ERK (p-ERK; top) compared with total expression of ERK (bottom). Bar graphs depict the quantitative densitometry analysis for Western blot densitometry data. Values are means ± SE of 4 independent experiments. ***P < 0.001, **P < 0.01 for comparison with control.

Article Snippet: Antibodies against phospho-c-Src (Tyr 416 ), phospho-cPLA 2 (Ser 505 ), phospho-p42MAPK (Try 202 )/p44MAPK (Try 204 ; ERK1/2), p42MAPK (Try 202 )/p44MAPK (Try 204 ), cPLA 2 , and c-Src were purchased from Cell Signaling Technology.

Techniques: Activity Assay, Transfection, Expressing, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: Use of a Glycolipid Inhibitor to Ameliorate Renal Cancer in a Mouse Model

doi: 10.1371/journal.pone.0063726

Figure Lengend Snippet: Representative immunoblots of tissue extracts from BALB/c mice fed vehicle (placebo plus RENCA cells/tumor) and 3 and 10 MPK of D-PDMP for 26 days and control kidney and corresponding densitometric scans are shown. A: D-PDMP dose dependently decreased the protein expression of LacCer synthase (β-1,4-GalT-V) (N = 3–6), in mouse renal cancer but somewhat increased the protein expression of UGCG (N = 2) in mouse renal cancer. B: Western immunoblots of markers for cell proliferation (p44MAPK, N = 3), angiogenesis (pAKT-1, N = 3; mTOR, N = 3 for placebo and 10 MPK) and house keeping protein GAPDH. D-PDMP treatment reduced the markers for cell proliferation and angiogenesis. Data were assessed by one-way ANOVA.

Article Snippet: Membranes were blocked in 3% milk, 1×TBST (0.05% Tween-20), incubated in primary antibodies against: β-1,4-GalT-V, Beta actin, UGCG (Santa Cruz Biotechnology), GAPDH (glyceraldehyde 3-phosphate dehydrogenase, US Biological), p44MAPK (Thr202 and Tyr204), p-AKT-1 (Thr308) (Cell Signaling Technologies), and mTOR (Sigma-Aldrich) in 1×TBS with 1∶200 dilution and 0.5% BSA, and incubated in secondary antibody, goat anti-rabbit IgG HRP conjugate (Sigma Aldrich) in 5% milk, 1×TBST with 1∶1000 dilution.

Techniques: Western Blot, Control, Expressing