Journal: Journal of Biological Chemistry

Article Title: Silencing of Insulin-like Growth Factor-binding Protein-2 in Human Glioblastoma Cells Reduces Both Invasiveness and Expression of Progression-associated Gene CD24

doi: 10.1074/jbc.m609567200

Figure Lengend Snippet: FIGURE 2. Modulation of IGF/IGF-IR signaling caused by knockdown of IGFBP-2. The IGFBP-2-knockdown cells (IGFBP-2 KD) and control cells were treated with the indicated concentrations of IGF-I or IGF-II in serum-free con- dition, and extents of phosphorylation of IGF-IR (p-IGF-IR), Akt (p-Akt), and ERK1/2 (p-Erk1/2) were analyzed by immunoblot and a representative result of U251 was shown. The band intensity was measured and the ratio of phos- phorylated protein to the corresponding total protein was calculated.

Article Snippet: The following primary antibodies were used: anti-human IGFBP-2 mouse monoclonal antibody (C-10; Santa Cruz Biotechnology, Santa Cruz, CA), anti- -actinmousemonoclonal antibody (AC-74; Sigma), anti-phosphoERK1/2 rabbit monoclonal antibody (Thr185/Tyr187, clone AW39;Upstate Cell Signaling Solutions, Lake Placid, NY), antiphospho-IGF-IR rabbit polyclonal antibody (Tyr1131), antiIGF-IR rabbit polyclonal antibody, anti-phospho-Akt mouse monoclonal antibody (Ser473), anti-Akt rabbit polyclonal antibody, anti-phospho-ERK1/2 rabbit monoclonal antibody (Thr185/Tyr187, clone AW39; Upstate Cell Signaling Solutions, Lake Placid, NY), anti-ERK1/2 rabbit polyclonal antibody, anticaspase-3, and cleaved capase-3 (Asp175) rabbit polyclonal antibodies, and anti-heat shock protein 70 rabbit polyclonal antibody (Cell Signaling Technology, Danvers, MA).

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

Journal: Nature metabolism

Article Title: Phagocytosis in the retina promotes local insulin production in the eye

doi: 10.1038/s42255-022-00728-0

Figure Lengend Snippet: a, Schematic for measuring C-peptide and Ins2 mRNA in fed and starved (10–14 h) conditions (left) and C-peptide cleavage during insulin synthesis (right). b, ELISA for C-peptide in blood and protein lysates from retina of fed (ad libitum) and starved mice. Y axis shows percentage of C-peptide found in fed mice. n = 5 mice for fed condition, n = 4 mice for starved. RT–PCR comparing Ins2 expression in RPE from fed or starved mice (right). n = 8 mice per condition. c, RT–PCR measuring Ins2 in RPE from control and Ins2 KO mice. n = 6 mice per genotype. d, Glucose tolerance assay (readout of insulin function) measuring circulating glucose levels over time in WT and Ins2 KO mice starved and then given glucose intraperitoneally (top). n = 4 mice per genotype. ELISA for C-peptide in blood of control and Ins2 KO mice shows no difference between genotypes (bottom). n = 4 mice per genotype. e, Schematic of the IPM (left). IPM was isolated from WT and Ins2 KO mice and the secreted C-peptide in IPM was measured by ELISA (middle). n = 4 mice per genotype. ELISA of C-peptide in lysates of RPE from WT and Ins2 KO mice (right). n = 4 mice WT, n = 3 Ins2 KO. f, Insulin receptor was immunoprecipitated from retina lysates of control and Ins2 KO mice, either fed or starved. InsR tyrosine phosphorylation was assessed using phospho-specific InsR antibodies via immunoblotting and quantified as the ratio of p-InsR to total InsR. Retina lysates were also probed for GLUT4 and quantified as the ratio of GLUT4 to actin (normalized to control). n = 4 mice for fed, n = 6 starved conditions. Plots are presented as in Fig. 1. *P ≤ 0.05,**P ≤ 0.01,***P ≤ 0.001, unpaired two-tailed t-test (b) and paired two-tailed t-test (f).

Article Snippet: Immunoprecipitation Equal amounts of protein lysate were incubated overnight at 4 °C with anti-insulin receptor-β antibody (10 g mg −1 lysate, Novus Biologicals, NBP2 12793) and pre-washed SureBeads Protein G Magnetic Beads (Bio-Rad).

Techniques: Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Isolation, Immunoprecipitation, Phospho-proteomics, Western Blot, Two Tailed Test

Journal: Nature metabolism

Article Title: Phagocytosis in the retina promotes local insulin production in the eye

doi: 10.1038/s42255-022-00728-0

Figure Lengend Snippet: a. RT–PCR of Ins1 fold change in mRNA of isolated RPE from control and Ins2 KO mice. n = 8 mice used for each genotype. b. Insulin receptor was immunoprecipitated from various tissue lysates of control and Ins2 KO mice that were starved overnight. Phosphorylation status of the InsR was probed using phospho-specific InsR antibodies via immunoblotting and quantified as the ratio of p-InsR to total InsR. n = 2 mice for control vs Ins2 KO. Plots are presented as in Fig. 1.

Article Snippet: Immunoprecipitation Equal amounts of protein lysate were incubated overnight at 4 °C with anti-insulin receptor-β antibody (10 g mg −1 lysate, Novus Biologicals, NBP2 12793) and pre-washed SureBeads Protein G Magnetic Beads (Bio-Rad).

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Control, Immunoprecipitation, Phospho-proteomics, Western Blot

Journal: Nature metabolism

Article Title: Phagocytosis in the retina promotes local insulin production in the eye

doi: 10.1038/s42255-022-00728-0

Figure Lengend Snippet: a. Schematic of streptozotocin (STZ) treatment. b. ELISA measuring C-peptide levels in blood of control, control STZ-treated, and Ins2 KO STZ-treated mice starved mice. N = 6 mice for control and N = 4 mice for STZ-treated Control and STZ-treated Ins2 KO. *p < .05, one-way ANOVA with Dunnet’s multiple comparisons test. c. Insulin receptor (InsR) was immunoprecipitated from retina tissue lysates of control, control STZ-treated, and Ins2KO STZ-treated mice, starved overnight (left) or control and control STZ-treated mice fed ad libitum (right). Phosphorylation status of the InsR was probed using phospho-specific antibodies (Tyr1150/1151) via immunoblotting and quantified as the ratio of p-InsR to total InsR, normalized to untreated WT mice. N = 3 mice for all conditions. *p < 05, one-way ANOVA with Tukey’s multiple comparison’s test. Plots are presented as in Fig. 1.

Article Snippet: Immunoprecipitation Equal amounts of protein lysate were incubated overnight at 4 °C with anti-insulin receptor-β antibody (10 g mg −1 lysate, Novus Biologicals, NBP2 12793) and pre-washed SureBeads Protein G Magnetic Beads (Bio-Rad).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Immunoprecipitation, Phospho-proteomics, Western Blot

Journal: Nature metabolism

Article Title: Phagocytosis in the retina promotes local insulin production in the eye

doi: 10.1038/s42255-022-00728-0

Figure Lengend Snippet: a, Representative image of three independent experiments showing immunofluorescence analysis with anti-rhodopsin (magenta) on retina sections 2 h after light onset (peak phagocytosis time) from fed and starved (10–14 h) mice. Sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (blue). RPE is outlined in white. b, Flow cytometry-based phagocytosis assay on isolated fixed and permeabilized RPE stained with rhodopsin, from fed or starved mice, obtained 2 h after light onset. Phagocytosis was measured as % of rhodopsin+ RPE within total RPE. n = 3 mice per condition. c, RT–PCR for Ins2 expression in RPE from fed mice at different times of the day. Lights on at 6:00 and lights off at 20:00. n = 8, 7, 7, 7, 3 and 3 mice used for times of 5:00, 6:00, 8:00, 15:00, 20:00 and 1:00, respectively. FC, fold change. d, InsR immunoprecipitated from retina lysates, at different times of day from overnight starved control and Ins2 KO mice, were probed using phospho-specific InsR antibodies. The same retina lysates were probed for GLUT4. Values were normalized to the average of control across all time points. n = 3 mice for each time point. e, Schematic of phagocytic receptors used in POS recognition (left). RT–PCR for Ins2 in RPE isolated from WT and MerTK KO or CD36 KO mice 2 h after light onset (right). n = 10 and 11 mice for WT versus MerTK KO and n = 6 and 5 mice for WT versus CD36 KO. f, Phagocytosis quantification using flow cytometry on isolated RPE from WT and MerTKCR mice (middle). n = 3 mice used per genotype. RT–PCR measuring Ins2 expression in isolated RPE from WT and MerTKCR (right). n = 14 and 10 mice for WT and MerTKCR, respectively. Plots are presented as in Fig. 1. *P ≤ 0.05,**P ≤ 0.01,***P ≤ 0.001, ****P ≤ 0.0001, one-way ANOVA (c), two-way ANOVA with Tukey’s multiple comparisons test (d), unpaired two-tailed t-test (e,f RT–PCR) and paired two-tailed t-test (f phagocytosis quantification).

Article Snippet: Immunoprecipitation Equal amounts of protein lysate were incubated overnight at 4 °C with anti-insulin receptor-β antibody (10 g mg −1 lysate, Novus Biologicals, NBP2 12793) and pre-washed SureBeads Protein G Magnetic Beads (Bio-Rad).

Techniques: Immunofluorescence, Flow Cytometry, Phagocytosis Assay, Isolation, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunoprecipitation, Control, Two Tailed Test

Journal: Nature metabolism

Article Title: Phagocytosis in the retina promotes local insulin production in the eye

doi: 10.1038/s42255-022-00728-0

Figure Lengend Snippet: a. Quantification of OS phagocytosis by the RPE 2 hours after light onset using immunohistochemistry. Quantification of phagocytosis was measured by the amount of Rhodopsin immunoreactivity in the RPE divided by pixels and presented as phagosomes per area on the y-axis (left). Quantification of the number of phagosomes in RPE was done by counting Rhodopsin puncta in the RPE (right). n = 3 mice used for each condition. *p ≤ .05 paired two-tailed t-test. b. Western blot against Rhodopsin on isolated RPE protein lysates from fed and starved mice at 8 am and 10 am (left). Right panel is quantification of the blot to evaluate POS degradation showing 10 am band intensity as a percent of 8 am (peak phagocytosis) band intensity (right). n = 2 mice used for each time point. c. Insulin receptor was immunoprecipitated from lysates of retina from control and MerTK KO mice that were starved overnight. The lysates were probed for InsR phosphorylation or GLUT4 levels by immunoblotting. C-peptide 2 levels were determined by ELISA. N = 6 mice for each condition. *p < .05 paired two-tailed t-test. d. Schematic of WT and cleavage-resistant ‘gain of function’ MerTKCR mice with altered cleavage sites indicated (left). e. Phagocytosis quantification of ingested photoreceptor outer segments using flow cytometry on isolated RPE stained with antibody against rhodopsin from Control and Ins2 KO mice two hours after light onset. n = 7 mice used for each genotype. Plots are presented as in Fig. 1.

Article Snippet: Immunoprecipitation Equal amounts of protein lysate were incubated overnight at 4 °C with anti-insulin receptor-β antibody (10 g mg −1 lysate, Novus Biologicals, NBP2 12793) and pre-washed SureBeads Protein G Magnetic Beads (Bio-Rad).

Techniques: Immunohistochemistry, Two Tailed Test, Western Blot, Isolation, Immunoprecipitation, Control, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining

Journal: International journal of molecular sciences

Article Title: Sex Modulates Response to Renal-Tubule-Targeted Insulin Receptor Deletion in Mice.

doi: 10.3390/ijms24098056

Figure Lengend Snippet: Figure 1. InsR knockout (KO) localization and degree in PAX8-targeted InsR KO mice—(A) β- galactosidase staining (indicating location of Cre-recombinase activity) in kidney cortex and medulla sections from mice that were carriers of both PAX8 and tet-O-Cre transgenes (left panels) or only PAX8 (right panels) and homozygously floxed for InsR; (B) Western blot of whole-kidney homogenates probed with InsR (β-subunit) antibody in WT and KO mice; (C) band densities of InsR Western blot (n = 6/genotype/sex, normalized to Ponceau staining); letters above bars indicate results of multiple comparison testing (MCT), with “A” significantly (p < 0.05) greater than “B”.

Article Snippet: Primary antibodies used for Western blotting were as follows: (1) InsR-β (A303–712A, polyclonal rabbit, Bethyl Laboratories, Montgomery, TX, USA); (2–4) α-, β-, and γ-ENaC (our own rabbit polyclonals) [18]; (5–7) NKCC2, AQP2, and NCC (our own rabbit polyclonals) [12]; (8) p-126-serine NKCC2 (rabbit polyclonal, a kind gift from Mark Knepper); (9) PEPCK (sc271019, rabbit polyclonal, Santa Cruz Biotechnology, Dallas, TX, USA); (10) FBP1 (109020, monoclonal rabbit, Abcam, Waltham, MA, USA); (11) G6PC (PAS-42541, polyclonal rabbit, Invitrogen, Waltham, MA, USA); (12) SGLT1 (NBP2-20338, polyclonal rabbit, NovusBio, Littleton, CO, USA); (13) SGLT2 (ab37296, polyclonal rabbit, Abcam), and (14) SNAT3 (14315, polyclonal rabbit, Proteintech, San Diego, CA, USA).

Techniques: Knock-Out, Staining, Activity Assay, Western Blot, Comparison

Journal: International journal of molecular sciences

Article Title: Sex Modulates Response to Renal-Tubule-Targeted Insulin Receptor Deletion in Mice.

doi: 10.3390/ijms24098056

Figure Lengend Snippet: Figure 2. Kidney weight, blood pressure, and heart rate in PAX8-targeted InsR KO mice—(A) mean arterial blood pressure (MAP); and (B) heart rate measured by tail cuff plethysmography; (C) kidney wet weight (n = 9–12/genotype/sex); p-values from two-way ANOVA (genotype; sex) are shown within each panel; letters above bars indicate results of MCT (only conducted when a main factor p < 0.05), with “A” assigned to the highest mean and all means not different from it, followed by “B”, etc. Bars with letters “not in common” are significantly different from each other, e.g., “AB” versus “C”.

Article Snippet: Primary antibodies used for Western blotting were as follows: (1) InsR-β (A303–712A, polyclonal rabbit, Bethyl Laboratories, Montgomery, TX, USA); (2–4) α-, β-, and γ-ENaC (our own rabbit polyclonals) [18]; (5–7) NKCC2, AQP2, and NCC (our own rabbit polyclonals) [12]; (8) p-126-serine NKCC2 (rabbit polyclonal, a kind gift from Mark Knepper); (9) PEPCK (sc271019, rabbit polyclonal, Santa Cruz Biotechnology, Dallas, TX, USA); (10) FBP1 (109020, monoclonal rabbit, Abcam, Waltham, MA, USA); (11) G6PC (PAS-42541, polyclonal rabbit, Invitrogen, Waltham, MA, USA); (12) SGLT1 (NBP2-20338, polyclonal rabbit, NovusBio, Littleton, CO, USA); (13) SGLT2 (ab37296, polyclonal rabbit, Abcam), and (14) SNAT3 (14315, polyclonal rabbit, Proteintech, San Diego, CA, USA).

Techniques:

Journal: International journal of molecular sciences

Article Title: Sex Modulates Response to Renal-Tubule-Targeted Insulin Receptor Deletion in Mice.

doi: 10.3390/ijms24098056

Figure Lengend Snippet: Figure 3. Reduced proximal tubule and collecting duct cell heights in InsR KO mice—(A) repre- sentative images of periodic acid–Schiff (PAS)-stained sections of kidney cortex (400×); (B) mean glomerular area; (C) mean PT and CCD cell height in WTM, KOM, WTF, and KOF mice; a two-way ANOVA (genotype; sex), followed by a multiple comparisons testing (MCT, Tukey’s), was conducted on the data (n = 9–12/genotype/sex); “A” is significantly higher than “B” by MCT; Glm—glomerulus; CCD—cortical collecting duct; PT—proximal tubule.

Article Snippet: Primary antibodies used for Western blotting were as follows: (1) InsR-β (A303–712A, polyclonal rabbit, Bethyl Laboratories, Montgomery, TX, USA); (2–4) α-, β-, and γ-ENaC (our own rabbit polyclonals) [18]; (5–7) NKCC2, AQP2, and NCC (our own rabbit polyclonals) [12]; (8) p-126-serine NKCC2 (rabbit polyclonal, a kind gift from Mark Knepper); (9) PEPCK (sc271019, rabbit polyclonal, Santa Cruz Biotechnology, Dallas, TX, USA); (10) FBP1 (109020, monoclonal rabbit, Abcam, Waltham, MA, USA); (11) G6PC (PAS-42541, polyclonal rabbit, Invitrogen, Waltham, MA, USA); (12) SGLT1 (NBP2-20338, polyclonal rabbit, NovusBio, Littleton, CO, USA); (13) SGLT2 (ab37296, polyclonal rabbit, Abcam), and (14) SNAT3 (14315, polyclonal rabbit, Proteintech, San Diego, CA, USA).

Techniques: Staining

Journal: The Journal of Physiology

Article Title: Isolating the effects of carbohydrate and lipid availability on exercise‐induced skeletal muscle signalling in males

doi: 10.1113/JP289864

Figure Lengend Snippet: Fold change of phosphorylation status and total protein content pre‐ to post‐exercise of INSR Tyr1150/1151 , Akt Ser473 , AS160 Thr642 , GLUT4, AMPK Thr172 , ACC Ser79 , PDK4, PDH Ser293 and HSL Ser660 ( B ) with representative blots ( A ). Data shown as the mean ± SD. Differences between FAST vs . CARB and CARB vs . NIACIN assessed using a paired t test ( n = 8 males).

Article Snippet: Membranes were washed again in TBS‐T and incubated in primary antibodies against phospho Ser 79 acetyl‐CoA carboxlyase (ACC) (Cell Signaling Technologies, Danvers, MA, USA), phospho Thr172 AMP‐dependent protein kinase (AMPK) (Cell Signaling Technologies), phospho‐Thr642 Akt substrate of 160 kDa (AS160) (Cell Signaling Technologies), phospho‐Tyr1150/1151 insulin receptor (INS) (Cell Signaling Technologies), phospho‐Ser 473 protein kinase B (Akt) (Cell Signaling Technologie), phospho‐Ser660 hormone‐sensitive lipase (HSL) (Cell Signaling Technologies), phospho‐Ser 293 pyruvate dehydrogenase (PDH) (Cell Signaling Technologies), vinculin (Cell Signaling Technologies), pyruvate dehydrogenase kinase 4 (PDK4) (Abgent, San Diego, CA, USA) and beta‐actin (Protein Technologies, Stockport, UK) overnight at 4°C.

Techniques: Phospho-proteomics

Journal: Journal of Tissue Engineering and Regenerative Medicine

Article Title: Improvement of hepatocyte engraftment by co‐transplantation with pancreatic islets in hepatocyte transplantation

doi: 10.1002/term.3170

Figure Lengend Snippet: Evaluation of the hepatocyte function by inhibition of candidate factors in coculture supernatant. The ammonia metabolic rate at 3 h in the coculture supernatant group ( n = 13) was significantly higher than that in the Basal medium group ( n = 13) (* p < 0.001). Although no inhibitory effects were seen in the Anti‐VEGF and GLP‐1 antagonist groups ( n = 5), the ammonia metabolic rate in the anti‐insulin group was significantly lower than that in the coculture supernatant group (* p < 0.001, n = 8). No significant difference was observed between the Basal medium and anti‐insulin groups

Article Snippet: Hepatocytes were cultured with basal medium (hepatocyte group, n = 13), coculture supernatant (coculture supernatant group, n = 13), coculture supernatant together with anti‐insulin receptor antibody (bs‐0290R, Bioss Inc) (Anti‐insulin group, n = 8), coculture supernatant together with 4 µg/ml anti‐VEGF antibody (ab9570, Abcam) (Anti‐VEGF group, n = 5), or coculture supernatant together with 50 ng/ml GLP‐1 antagonist (ab141101, Abcam) (GLP‐1 antagonist group, n = 5).

Techniques: Inhibition