Journal: International Journal of Molecular Sciences

Article Title: Paederoside Promotes Longevity and Fitness in C. elegans Through Ubiquitination and Degradation of DAF-2/IGF1R, Activating DAF-16/FOXO and SKN-1/NRF2 Transcription Factors

doi: 10.3390/ijms27052248

Figure Lengend Snippet: ( a ) Molecular docking outcomes. Western blot analysis of ( b ) ubiquitination, ( c ) IGF1R, ( d ) CoIP and ( f ) key proteins of the IIS pathway. ( e ) FOXO3 nuclear translocation assay. The levels of statistical significance were set at * p < 0.05, ** p < 0.01, and **** p < 0.0001, ns and nd indicates no statistical significance.

Article Snippet: The antibodies used in this study were as follows: β-actin (HUABIO, Hangzhou, China, 1:20,000, HA722023); β-Tubulin (HUABIO, Hangzhou, China, 1:20,000, EM0103); IGF1R (abcam, Cambridge, MA, USA, 1:1000, ab182408, d,f); IGF1R (Proteintech Group, Wuhan, China, 1:1000, 21707-1-AP, c); ubiquitin (PTM bio, Hangzhou, China, 1:1000, PTM-7228); AKT3 (Absea Biotechnology, Suzhou, China, 1:1000, OC475); pAKT (Absea Biotechnology, Suzhou, China, 1:1000, RC4352); FOXO3 (abmart, Cambridge, MA, USA, 1:1000, PA5461S); and anti-Rabbit IgG-HRP (HUABIO, Hangzhou, China, 1:20,000, HA1001).

Techniques: Western Blot, Ubiquitin Proteomics, Nuclear Translocation Assay

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Effects of Pas2r12 or Pas2r12–cargo protein complex-mediated stimulation on INSR/IGF1R and ERK1/2. HEK293 cells were pretreated with dimethyl sulfoxide (linsitinib [−]) or with linsitinib (linsitinib [+]) and subsequently stimulated with Pas2r12, Pas2r12–EGFP, Pas2r12–IgG, or insulin for 2 min ( A – C ) or 10 min ( D – F ). Panels A and D show representative Western blot images. Panels B and E display the phosphorylation levels of INSR/IGF1R (pINSR/pIGF1R); panels C and F show the phosphorylation levels of ERK1/2 (pERK1/2); all values were normalized to GAPDH. Phosphorylation levels were analyzed and compared with the solvent control (DMEM only, linsitinib [−]) using Student’s t -test. For each treatment condition, linsitinib (+) was compared with the corresponding linsitinib (−) condition. Statistically significant differences compared with the control are indicated on the bar graphs; significant differences between linsitinib (+) and linsitinib (−) treatments are marked by horizontal lines. Statistical comparisons were performed against control cells using Student’s t -test. Error bars indicate the standard error of the mean (SEM). * p < 0.05 and ** p < 0.01. N = 4.

Article Snippet: Transfection was performed using Lipofectamine 3000 with 5 μg of the IGF1R CRISPR Activation Plasmid (sc-400084-ACT; Santa Cruz Biotechnology).

Techniques: Western Blot, Phospho-proteomics, Solvent, Control

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Effect of IGF1R and INSR knockdown on the Pas2r12-mediated cytosolic delivery of EGFP. Western blot analyses ( A – C ) and confocal laser scanning microscopy images ( D , E ). Graphs B and C show IGF1R and INSR expression levels, respectively, normalized to GAPDH. Cells analyzed with confocal laser scanning microscopy in panel A were analyzed by Western blot ( A – C ). ( D ) Pas2r12-mediated cytosolic delivery of EGFP in knockdown cells, with EGFP fluorescence shown in green, and ( E ) percentage of cells exhibiting cytosolic EGFP delivery. Scale bars represent 20 μm. Statistical comparisons were performed against siNC cells using Student’s t -test. Error bars indicate the standard error of the mean (SEM). * p < 0.05, *** p < 0.001. N = 4.

Article Snippet: Transfection was performed using Lipofectamine 3000 with 5 μg of the IGF1R CRISPR Activation Plasmid (sc-400084-ACT; Santa Cruz Biotechnology).

Techniques: Knockdown, Western Blot, Confocal Laser Scanning Microscopy, Expressing, Fluorescence

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Assessment of IGF1R overexpression. ( A ) Verification of IGF1R expression levels in HEKI cells. ( B ) Relative levels of IGF1R expression normalized to GAPDH based on the data in panel A. Values for IGF1R/GAPDH are expressed compared with parental HEK293 cells (set to 1). Statistical comparisons were performed against HEK293 cells using Student’s t -test. Error bars indicate the standard error of the mean (SEM). * p < 0.05, *** p < 0.001. N = 3. ( C ) Subcellular localization of IGF1R is shown in green; nuclei were counterstained with Hoechst 33342 (blue). Scale bars represent 20 μm.

Article Snippet: Transfection was performed using Lipofectamine 3000 with 5 μg of the IGF1R CRISPR Activation Plasmid (sc-400084-ACT; Santa Cruz Biotechnology).

Techniques: Over Expression, Expressing

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Effects of Pas2r12 or Pas2r12–cargo protein complex stimulation on INSR/IGF1R in IGF1R-overexpressing cells. ( A ) HEKI#66 cells pretreated with DMSO (linsitinib [−]) or linsitinib (linsitinib [+]) were stimulated with Pas2r12, Pas2r12–EGFP, or insulin for 2 min. Level of phosphorylated INSR/IGF1R (pINSR/pIGF1R) was assessed by Western blot. ( B ) Relative levels of pINSR/pIGF1R, normalized to GAPDH, corresponding to the data in panel A. Phosphorylation levels were analyzed using Student’s t -test. Each treatment condition was compared with the solvent-treated HEKI#66 control (linsitinib [−]), and, for each treatment, phosphorylation levels in the linsitinib (−) and linsitinib (+) conditions were also compared. Statistically significant differences compared with the solvent-treated control are indicated on the bar graphs, and significant differences between linsitinib (+) and linsitinib (−) treatments are marked by horizontal lines. Error bars indicate the standard error of the mean (SEM). * p < 0.05, *** p < 0.001. N = 3.

Article Snippet: Transfection was performed using Lipofectamine 3000 with 5 μg of the IGF1R CRISPR Activation Plasmid (sc-400084-ACT; Santa Cruz Biotechnology).

Techniques: Western Blot, Phospho-proteomics, Solvent, Control

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Effect of IGF1R overexpression on the Pas2r12-mediated cytosolic delivery of EGFP. ( A ) Representative confocal images showing the cellular uptake of Pas2r12–EGFP in IGF1R-overexpressing (HEKI) cells. Scale bars represent 20 μm. ( B ) Relative levels of the cytosolic delivery efficiency of EGFP in HEKI cells compared with parental HEK293 cells (set to 100%). Merged images show EGFP fluorescence (green) and differential interference contrast. Statistical comparisons were performed against HEK293 cells using Student’s t -test. Error bars indicate the standard error of the mean (SEM). ** p < 0.01. N = 3.

Article Snippet: Transfection was performed using Lipofectamine 3000 with 5 μg of the IGF1R CRISPR Activation Plasmid (sc-400084-ACT; Santa Cruz Biotechnology).

Techniques: Over Expression, Fluorescence

Journal: Pharmaceuticals

Article Title: Role of the Insulin Receptor in Mediating Cytosolic Delivery of Proteins by a Modified Cell-Penetrating Peptide

doi: 10.3390/ph18121885

Figure Lengend Snippet: Effects of Pas2r12 or Pas2r12–cargo protein complex stimulation on INSR/IGF1R in INSR-overexpressing cells. ( A ) IN#1 cells pretreated with DMSO (linsitinib [−]) or linsitinib (linsitinib [+]) were stimulated with Pas2r12, Pas2r12–EGFP, or insulin for 2 min. Phosphorylation of INSR/IGF1R (pINSR/pIGF1R) was assessed by Western blot. ( B ) Relative levels of pINSR/pIGF1R were normalized to GAPDH, corresponding to the data in panel A. Phosphorylation levels were analyzed using Student’s t -test. Each treatment condition was compared with the solvent-treated IN#1 control (linsitinib [−]), and for each treatment, phosphorylation levels in the linsitinib (−) and linsitinib (+) conditions were also compared. Statistically significant differences compared with the solvent-treated control are indicated on the bar graphs, and significant differences between linsitinib (+) and linsitinib (−) treatments are marked by horizontal lines. Error bars indicate the standard error of the mean (SEM). * p < 0.05, ** p < 0.01, and *** p < 0.001. N = 3.

Article Snippet: Transfection was performed using Lipofectamine 3000 with 5 μg of the IGF1R CRISPR Activation Plasmid (sc-400084-ACT; Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Western Blot, Solvent, Control

Journal: Breast Cancer Research : BCR

Article Title: erbB3 recruitment of insulin receptor substrate 1 modulates insulin-like growth factor receptor signalling in oestrogen receptor-positive breast cancer cell lines

doi: 10.1186/bcr3018

Figure Lengend Snippet: Western blot analysis . (a) Total and phosphorylated insulin-like growth factor type I receptor (IGF-IR), insulin receptor substrate 1 (IRS-1), erbB3, Akt and extracellular-signal regulated kinase 1/2 (ERK1/2) protein expression following incubation of MCF-7 and T47D cells in medium containing either the specific IGF-IR/IR tyrosine kinase inhibitor 4-anilino-5-bromo-2-[4-(2-hydroxy-3-( N , N -dimethylamino)propoxy)anilino]pyrimidine (ABDP) (1 μM) or appropriate vehicle control for 24 hours and subsequently challenged with increasing concentrations of heregulin β1 (HRGβ1) (0.1 to 10 ng/ml) or vehicle control for 5 minutes. Densitometric analysis of phosphorylated Akt and ERK1/2 protein levels in (b) MCF-7 and (c) T47D cells treated with HRGβ1 in the absence and presence of ABDP. The results are expressed as the means ± standard errors of the mean of at least three separate experiments from the 1 ng/ml HRGβ1-primed groups. (d) Western blot analysis of total IGF-IR, erbB3 and IRS-1 expression following immunoprecipitation (IP) with total IRS-1 antibody in MCF-7 and T47D cells incubated for 24 hours in medium containing ABDP (1 μM) or vehicle control. * P ≤ 0.05 versus HRGβ1 for both cell lines.

Article Snippet: To examine the effects of pharmacological blockade of IGF-IR, cells were incubated in phenol red-free (white) RPMI medium supplemented with 5% FCS and either the IGF-IR/IR tyrosine kinase inhibitor 4-anilino-5-bromo-2-[4-(2-hydroxy-3-( N , N -dimethylamino)propoxy)anilino]pyrimidine (ABDP) (1 μM in dimethyl sulphoxide, AstraZeneca, Macclesfield, UK) [ ] or appropriate vehicle control for 1 to 2 days.

Techniques: Western Blot, Expressing, Incubation, Control, Immunoprecipitation

Journal: Breast Cancer Research : BCR

Article Title: erbB3 recruitment of insulin receptor substrate 1 modulates insulin-like growth factor receptor signalling in oestrogen receptor-positive breast cancer cell lines

doi: 10.1186/bcr3018

Figure Lengend Snippet: Western blot analysis . (a) Total and phosphorylated insulin-like growth factor type I receptor (IGF-IR), insulin receptor substrate 1 (IRS-1), Akt, extracellular signal-regulated kinase 1/2 (ERK1/2) and β-actin protein expression following incubation of MCF-7 and T47D cells in medium containing either lipid and control siRNA (C si) mix (100 nM) or lipid and IRS si mix (100 nM) in the absence or presence of 4-anilino-5-bromo-2-[4-(2-hydroxy-3-( N , N -dimethylamino)propoxy)anilino]pyrimidine (ABDP) (1 μM) for 2 days and subsequently challenged with heregulin β1 (HRGβ1) (10 ng/ml) for 5 minutes. Data are representative of at least three separate experiments. The effects of 4-day treatment with HRGβ1 (10 ng/ml), ABDP (0.1 μM) or a combination of HRGβ1 and ABDP on the growth of (b) MCF-7 cells or (c) T47D cells. The results are expressed as the means ± standard errors of the mean of triplicate wells and are representative of at least three separate experiments. * P ≤ 0.01 versus control, † P ≤ 0.001 versus ABDP.

Article Snippet: To examine the effects of pharmacological blockade of IGF-IR, cells were incubated in phenol red-free (white) RPMI medium supplemented with 5% FCS and either the IGF-IR/IR tyrosine kinase inhibitor 4-anilino-5-bromo-2-[4-(2-hydroxy-3-( N , N -dimethylamino)propoxy)anilino]pyrimidine (ABDP) (1 μM in dimethyl sulphoxide, AstraZeneca, Macclesfield, UK) [ ] or appropriate vehicle control for 1 to 2 days.

Techniques: Western Blot, Expressing, Incubation, Control