antibody anti-ppdk1 s241  (Cell Signaling Technology Inc)

Journal: Cellular and Molecular Life Sciences

Article Title: Non-peptidyl small molecule, adenosine, 5′-Se-methyl-5′-seleno-, 2′,3′-diacetate, activates insulin receptor and attenuates hyperglycemia in type 2 diabetic Lepr db/db mice

doi: 10.1007/s00018-019-03249-4

Figure Lengend Snippet: Activation of Insr/Akt/AS160 signaling in both differentiated mouse C2C12 cells and skeletal muscle of Lepr db/db mice by NPC43 and cooperative action of NPC43 and insulin in the stimulation of glucose uptake in differentiated C2C12 cells. a Activation of Insr and stimulation of Akt and AS160 phosphorylation in differentiated mouse C2C12 (skeletal muscle) cells by NPC43. Completely differentiated C2C12 cells were serum-starved overnight, treated without (Control) or with NPC43 (7.6 μM), insulin (0.2 μM) or both in serum-free DMEM media at 37 °C for 5 (top panel) or 60 (bottom panel) minutes and then subjected to Western blot analysis (using 8 μg protein, triplicates/group). Quantitative data of the expression levels of these proteins detected in Western blots are shown in Online Resource 7. b Activation of Insr and stimulation of Akt and AS160 phosphorylation in the skeletal muscle of Lepr db/db mice by NPC43. Five Lepr db/db mice at postnatal day 38 were i.p. injected with 0.2% (v/v) DMSO/saline or NPC43 (0.136 mg/kg BW) daily for 52 days. Gastrocnemius from these mice was collected and subjected to Western blot analysis (using 100 μg protein/mouse) of pInsrβ Y1146 (activated Insr), pPdk1 S241 and pAkt T308 or immunoprecipitation (IP, using 400 μg protein/mouse) with a specific AS160 monoclonal antibody followed by Western blot analysis of pAS160 S588. Quantitative data for protein expression of the above Insr signaling molecules are shown in Online Resource 9. c Effects of insulin and NPC43 on glucose uptake in differentiated mouse C2C12 cells. C2C12 cells (5000 cells/96-well) were cultured in 10% FBS/DMEM media for 5 days and then differentiated using 0.5% horse serum/DMEM media for 7 days. These differentiated C2C12 cells were pretreated without or with NPC43 (3.8 or 7.6 μM) in serum- and glucose-free DMEM media overnight and then treated without (basal) or with insulin (0.2 μM), NPC43 (3.8 and 7.6 μM) or both insulin and NPC43 in serum- and glucose-free DMEM media at 37 °C for 1.5 h followed by incubation with 1 mM 2DG at room temperature for 30 min. Luminescence of up-taken glucose (i.e., 2DG) was determined using a luminometer. Data are presented as mean ± SD of indicated number of replicates per group. ** P < 0.01, *** P < 0.001, vs. the basal group (Student’s t test). Percentage in parentheses in the bar graph refers to the mean percentage of increased glucose uptake in that group when compared to the basal group

Article Snippet: Primary antibodies against pINSRβ at Y1146 (Cat #3021) or Y1150/1151 (Cat #3024), INSRβ (Cat #3025), pPDK1 at S241 (Cat #3438), PDK1 (Cat #3062), pAKT at T308 (Cat #2965) or S473 (Cat #4060), AKT (Cat #4691), pFOXO1 at T24 (Cat #9464) or S256 (Cat #9461), FOXO1 (Cat #2880), pAS160 at S588 (Cat #8730), AS160 (Cat #2670), pIGF1Rβ at Y1131 (Cat #3021), IGF1Rβ (Cat #3027), ACTB (Cat #8457) and β-tubulin (Cat #2146) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Western Blot, Expressing, Injection, Immunoprecipitation, Cell Culture, Incubation