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: Membranes were incubated with primary monoclonal antibodies against phospho-insulin receptor β (p-IRβ; clone 10C3, sc-81500; Santa Cruz Biotechnology, Dallas, TX, USA), INSR (MAA895Hu21; Cloud-Clone Corp., Houston, TX, USA), phospho-ERK1/2 (sc-7383; Santa Cruz Biotechnology), ERK1/2 (sc-514302; Santa Cruz Biotechnology), and IGF-1 receptor (MAB659Hu22; Cloud-Clone Corp).

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: Membranes were incubated with primary monoclonal antibodies against phospho-insulin receptor β (p-IRβ; clone 10C3, sc-81500; Santa Cruz Biotechnology, Dallas, TX, USA), INSR (MAA895Hu21; Cloud-Clone Corp., Houston, TX, USA), phospho-ERK1/2 (sc-7383; Santa Cruz Biotechnology), ERK1/2 (sc-514302; Santa Cruz Biotechnology), and IGF-1 receptor (MAB659Hu22; Cloud-Clone Corp).

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: 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: Membranes were incubated with primary monoclonal antibodies against phospho-insulin receptor β (p-IRβ; clone 10C3, sc-81500; Santa Cruz Biotechnology, Dallas, TX, USA), INSR (MAA895Hu21; Cloud-Clone Corp., Houston, TX, USA), phospho-ERK1/2 (sc-7383; Santa Cruz Biotechnology), ERK1/2 (sc-514302; Santa Cruz Biotechnology), and IGF-1 receptor (MAB659Hu22; Cloud-Clone Corp).

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: Assessment of INSR overexpression. ( A ) Verification of INSR expression levels in INSR-overexpressing (IN) cells. ( B ) Relative levels of INSR expression normalized to GAPDH, based on the data in panel A. The value for INSR/GAPDH in parental HEK293 cells was set to 1.0 for comparison. 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 and ** p < 0.01. N = 3. ( C ) Subcellular localization of INSR in IN cells. Green indicates INSR, and blue indicates nuclei. Scale bars represent 20 μm.

Article Snippet: Membranes were incubated with primary monoclonal antibodies against phospho-insulin receptor β (p-IRβ; clone 10C3, sc-81500; Santa Cruz Biotechnology, Dallas, TX, USA), INSR (MAA895Hu21; Cloud-Clone Corp., Houston, TX, USA), phospho-ERK1/2 (sc-7383; Santa Cruz Biotechnology), ERK1/2 (sc-514302; Santa Cruz Biotechnology), and IGF-1 receptor (MAB659Hu22; Cloud-Clone Corp).

Techniques: Over Expression, Expressing, Comparison

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: Membranes were incubated with primary monoclonal antibodies against phospho-insulin receptor β (p-IRβ; clone 10C3, sc-81500; Santa Cruz Biotechnology, Dallas, TX, USA), INSR (MAA895Hu21; Cloud-Clone Corp., Houston, TX, USA), phospho-ERK1/2 (sc-7383; Santa Cruz Biotechnology), ERK1/2 (sc-514302; Santa Cruz Biotechnology), and IGF-1 receptor (MAB659Hu22; Cloud-Clone Corp).

Techniques: Phospho-proteomics, Western Blot, 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 INSR overexpression on the Pas2r12-mediated cytosolic delivery of EGFP. ( A ) Representative confocal images showing cellular uptake of Pas2r12–EGFP in INSR-overexpressing (IN) cells. Merged images show EGFP fluorescence (green), Hoechst 33342 nuclear staining (blue), and differential interference contrast. Scale bars represent 20 μm. ( B ) Relative levels of the cytosolic delivery efficiency of EGFP in IN cells compared with parental HEK293 cells (set to 100%). 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. N = 3.

Article Snippet: Membranes were incubated with primary monoclonal antibodies against phospho-insulin receptor β (p-IRβ; clone 10C3, sc-81500; Santa Cruz Biotechnology, Dallas, TX, USA), INSR (MAA895Hu21; Cloud-Clone Corp., Houston, TX, USA), phospho-ERK1/2 (sc-7383; Santa Cruz Biotechnology), ERK1/2 (sc-514302; Santa Cruz Biotechnology), and IGF-1 receptor (MAB659Hu22; Cloud-Clone Corp).

Techniques: Over Expression, Fluorescence, Staining