Concentration dependent Sorbitol-induced activation of p38 MAP kinase. Various concentrations of sorbitol treated retinal samples were subjected to Western blot analysis with anti-phospho-p38MAP kinase (A) and anti-p38 MAP kinase (B) antibodies.

Sorbitol-induced activation of Akt isoforms. Various concentrations of sorbitol treated retinal samples were subjected to Western blot analysis with anti-pAkt (S473) (A) and anti-Akt (B) antibodies. Retinas were incubated in culture and treated with either insulin (1 μM) or sorbitol (3M). Retinas were lysed and the proteins were subjected to immunoprecipitation with anti-Akt1, anti-Akt2 and Anti-Akt3 antibodies followed by Western blot analysis with anti-pAkt (S473) (C and D) antibody. Input, 30 μg of retina lysates stimulated with either presence or absence of insulin or sorbitol.

PI3K-independent activation of Akt. Rat retinas were pre-incubated in DMEM medium with or without the PI3K inhibitor LY294002 (50 μM) for 30 min prior to either 1μM insulin or 3 M sorbitol. Thirty micrograms of retina lysate were subjected to Western blot analysis with anti-phospho-Akt (Ser 473) (A). The blot was stripped and reprobed with anti-Akt (B) and anti-actin (C) antibodies. PI3K activation is independent of p38 MAP kinase activation. Rat retinas were pre-incubated in DMEM medium with or without the PI3K inhibitor LY294002 (50 μM) or MAP kinase inhibitor SB203580 (50 μM) for 30 min prior to the treatment of either 1μM insulin or 3 M sorbitol. Thirty micrograms of retina lysates were subjected to Western blot analysis with anti-phospho-p38 MAP kinase(D). The blot was stripped and reprobed with anti-p38 MAP kinase (E) and anti-actin (F) antibodies.

Tyrosine kinase induced-activation of Akt. Rat retinas were pre-incubated in DMEM medium with or without 100 μM genestin or HNMP3 or PP1 or PP2 or PP3 for 30 min prior to the treatment of 1 M sorbitol for 30 min. Thirty micrograms of retina lysates were subjected to Western blot analysis with anti-pAkt (S473) (A) anti-Akt (B) and anti-actin (C) antibodies.

Sorbitol-induced activation of phosphatase activity. Retinal proteins from control and sorbitol-treated (1.0 or 3.0 M) or untreated organotypic cultures were subjected to either Western blotting analysis with anti-pAkt (B) and anti-Akt (C) antibodies or measured the phosphatase activity using pNPP as substrate (A). Open bars represent the activity in the presence of 1mM sodium vanadate. Purified GST-PTP1B (5μg) was used as positive control. Data are Data mean ± SD, n= three independent experiments 3, *p<0.001.

PTP1B dephosphorylates the sorbitol-induced tyrosine phosphorylated proteins in vitro. Sorbitol-treated retinal proteins were incubated with either wild type PTP1B or catalytically inactive PTP1B (D181A) for 15 min at 37 °C. At the end of the reaction, proteins were subjected to SDS-PAGE followed by Western blot analysis with anti-PY99 antibody (A). The blot was reprobed with anti-GST (B) and anti-actin (C) antibodies.

Sorbitol-induced activation of IR and IGF-1R. Retinal proteins from control and 1.0 M sorbitol-treated organotypic cultures were immunoprecipitated with anti-IRß antibody followed by Western blot analysis with anti-PY99 antibody (A). The blots were stripped and reprobed with anti-IRß antibody to ensure equal amounts of IR in each immunoprecipitate (B). Densities were calculated from the immunoblots and the results are expressed as percentage of PY99/IR. Absence of sorbitol was taken as 100 percent (C). Data mean ± SD, n=3, *p<0.001. Sorbitol-induced activation of IGF 1R. Retinal proteins from the control and 1.0 M sorbitol-treated organotypic cultures were immunoprecipitated with anti-IGF 1R antibody followed by Western blot analysis with anti-PY99 antibody (D). The blots were stripped and reprobed with anti-IGF 1R antibody to ensure equal amount of IGF 1R in each immunoprecipitate (E). Densities were calculated from the immunoblots and the results are expressed as percentage of PY99/IR. Absence of sorbitol was taken as 100 percent (F). Data mean ± SD, n=3, *p<0.001.

Sorbitol-induced activation insulin receptor associated PI3K activity. Retinas were cultured in DMEM and treated with various concentrations of sorbitol (0-3 M) for 30 min at 37 °C. TLC autoradiogram of PI3K activity measured in anti-IRβ immunoprecipitates of retinas using PI-4,5-P₂ and [γ³²P]ATP as substrates (A). The radioactive spots of PI-3,4,5-P₃ were scraped from the TLC plate and counted (B).

Sorbitol-induced activation of Akt is calcium-independent. Rat retinas were pre-incubated in DMEM medium with or without 15 μM BAPTA for 30 min prior to the treatment of 1 M sorbitol for additional 30 min. Thirty micrograms of retina lysates were subjected to Western blot analysis with anti-pAkt (S473) (A) anti-Akt (B), anti-actin (C) and anti-PY99 (D) antibodies.

Cholesterol-depletion results in the activation of Akt. Rat retinas were incubated in DMEM medium with or without MCD prior to the treatment of 1 M sorbitol for 30 min. Thirty micrograms of retina lysates were subjected to Western blot analysis with anti-pAkt (S473) (A) and anti-Akt (B) antibodies.

Sorbitol-induced tyrosine phosphorylation of several retinal proteins. Retinas were cultured in DMEM in the presence or the absence of either 1 μM insulin or various concentrations of sorbitol for 30 min at 37 °C. Thirty micrograms of retinal proteins were subjected to Western blot analysis with anti-PY 99 antibody (A). The blot was reprobed with the anti-actin antibody to ensure equal amount of protein in each lane (B). ROS and non-ROS proteins were subjected to Western blot analysis with the anti-PY99 antibody (C).

Interaction of Cbl with p85 subunit of PI3K. Sorbitol (1.0 M) treated or untreated retinal lysates were subjected to GST-pull down assay with GST-p85 full-length fusion protein followed by Western blot analysis with anti-Cbl antibody (A). Sorbitol treated and untreated retinal lysates were subjected to immunoprecipitation with anti-Cbl antibody followed by either Western blot analysis with anti-PY99 antibody (B) or measured the Cbl associated PI3K activity (C). Sorbitol treated and untreated retinal lysates were immunoprecipitated with anti-ubiquitin antibody followed by Western blot analysis with anti-p85 subunit of PI3K antibody (D).