Journal: Oncology Letters

Article Title: The cytotoxicity of gefitinib on patient‑derived induced pluripotent stem cells reflects gefitinib‑induced liver injury in the clinical setting

doi: 10.3892/ol.2023.14108

Figure Lengend Snippet: EGFR expression, and the phosphorylation following gefitinib treatment in the clones of patient-derived iPSCs or iPSC-heps from the N or T group. Western blot analyses of (A) iPSCs and (B) iPSC-heps from the N or T group. EGFR expression was detected with an anti-EGFR antibody. Densitometric analysis was performed using ImageJ software for (C) iPSCs and (D) iPSC-heps. (E) iPSCs were cultured with gefitinib at indicated concentrations (0, 3, 6 µM) for two days. The cell lysates were subjected to western blot analysis. EGFR and pEGFR expressions were detected with their respective antibodies (Upper panel). GAPDH was used as a loading control. (F) Densitometric analysis (n=3) was performed using ImageJ software (Lower panel). The values represented mean ± SD. *P<0.05, Dunnett's test. T, toxicity; N, no toxicity; EGFR, epidermal growth factor receptor; pEGFR, phosphorylated EGFR; iPSCs, induced pluripotent stem cells; iPSC-heps, iPSC-hepatocytes.

Article Snippet: Anti-EGFR antibodies (sheep polyclonal, Upstate ® , Merck, Darmstadt, DE), phospho-EGFR (Y1068) [mouse monoclonal (m), Abcam, Cambridge, UK], GAPDH (m, MBL, Nagoya, Japan), human albumin (goat polyclonal, Bethyl Laboratories, Montgomery, TX), α-fetoprotein [rabbit monoclonal (r, mAb), Abcam, Cambridge, UK], and hepatocyte nuclear factor 4 alpha (HNF4α) (r, mAb, Abcam, Cambridge, UK) were used for our experiments.

Techniques: Expressing, Phospho-proteomics, Clone Assay, Derivative Assay, Western Blot, Software, Cell Culture, Control

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: Bound proteins were boiled in 25 μl Laemmli's sample buffer (2×) for 5 min and resolved by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, blocked in 5% nonfat dry milk in PBS-Tween 20 (0.1%) for 1 h at room temperature, and subsequently incubated with anti-EGFR sheep polyclonal antibody (1:1,000 dilution, Sigma) overnight at 4°C.

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: Bound proteins were boiled in 25 μl Laemmli's sample buffer (2×) for 5 min and resolved by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, blocked in 5% nonfat dry milk in PBS-Tween 20 (0.1%) for 1 h at room temperature, and subsequently incubated with anti-EGFR sheep polyclonal antibody (1:1,000 dilution, Sigma) overnight at 4°C.

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 induces binding of EGFR with the SH2 domain of c-Src 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), AA (15 μmol/l), ETYA (15 μmol/l), or ANG II (1 μmol/l) for 5 min or plasmids encoding β1 and γ2 (5 μg each) for 24 h. At these treatments, interaction of the EGFR with glutathione-S-transferase (GST)-c-Src SH2 and GST-c-Src SH3 was performed by pull-down of GST fusion protein (IP) and immunoblotted (IB) with anti-EGFR antibody (top). The membrane was stripped and reprobed with an anti-c-Src monoclonal antibody (bottom). Comparable amounts of proteins were detected in immunoprecipitates of all the above experiments. Quantitation of EGFR phosphorylation was determined semiquantitively by densitometric scanning of each Western blot. Bar graphs depict the quantitative densitometry analysis for Western blot densitometry data. Values are means ± SE of 4 independent experiments. **P < 0.01 for comparison with control.

Article Snippet: Bound proteins were boiled in 25 μl Laemmli's sample buffer (2×) for 5 min and resolved by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, blocked in 5% nonfat dry milk in PBS-Tween 20 (0.1%) for 1 h at room temperature, and subsequently incubated with anti-EGFR sheep polyclonal antibody (1:1,000 dilution, Sigma) overnight at 4°C.

Techniques: Binding Assay, Plasmid Preparation, Transfection, Quantitation Assay, Western Blot

Journal: Growth factors (Chur, Switzerland)

Article Title: EGFR ligands exhibit functional differences in models of paracrine and autocrine signaling

doi: 10.3109/08977194.2011.649918

Figure Lengend Snippet: Table I

Article Snippet: In some cases, a parallel blot was probed with an anti-EGFR sheep polyclonal antibody (Capralogics, Gilbertville, MA, USA).

Techniques: DNA Synthesis