Journal: Neoplasia (New York, N.Y.)

Article Title: Activation of HER3 Interferes with Antitumor Effects of Axl Receptor Tyrosine Kinase Inhibitors: Suggestion of Combination Therapy 1

doi: 10.1016/j.neo.2014.03.009

Figure Lengend Snippet: HER3 activation is a common feedback mechanism of Axl inhibitors, and MPCD84111 blocks phosphorylation of HER3 in contrast to BMS777607. (A) Inhibition of Axl phosphorylation was determined 1 hour posttreatment with BMS777607, MPCD84111, and R428 by p-Tyr–Axl ELISA in NIH/3T3-Axl cells. IC50 values were calculated by four-parameter log curve fit. Axl kinase activity was inhibited in a dose-dependent manner, with an IC50 value of 0.006 μM for BMS777607, 0.027 μM for MPCD84111, and 0.043 μM for R428. (B) Axl Inhibitors induce HER3 expression. Western blot analysis of MDA-MB231 cells treated with 10 μM BMS777607, MPCD84111, and R428 for 24 hours. BMS777607 and R428 caused an increase in pHER3 Y1289 after 24 hours of treatment in contrast to MPCD84111. Treatment with all three Axl inhibitors leads to a six- to 7.5-fold up-regulation of HER3 protein levels. The diagrams show the densitometric analysis of Western blots for pHER3 Y1289 and HER3. Mean values and SEM are shown (n = 3). (C) MPCD84111 blocks phosphorylation of HER3 in contrast to BMS777607. Western blot analysis of MDA-MB231 cells treated with 1 μM BMS777607 or MPCD84111 up to 48 hours is shown. BMS777607 caused an increase in pHER3 Y1289 after 6 hours of treatment in contrast to MPCD84111. Both inhibitors induced a significant increase in protein expression of HER3 after 16 hours of treatment. (D) The kinase selectivity profile against a panel of 36 human kinases proves HER2 as a direct target of MPCD84111. The selectivity profiling was performed in triplicate at a compound concentration of 10 μM. The plots indicate the percentages of inhibition for each individual kinase.

Article Snippet: The Pan AKT-specific ELISA kit (R&D Systems GmbH, Wiesbaden-Nordenstadt, Germany, No. DYC887B-5) was used for quantification of phospho-AKT S473 according to the manufacturer's protocol with the following modifications: After the incubation with a biotinylated phospho-AKT (S473) panspecific detection antibody (1:180), we used an alkaline phosphatase–conjugated streptavidin SA110 (Millipore, Schwalbach, Germany) (1:4000) at room temperature.

Techniques: Activation Assay, Phospho-proteomics, Inhibition, Enzyme-linked Immunosorbent Assay, Activity Assay, Expressing, Western Blot, Concentration Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Activation of HER3 Interferes with Antitumor Effects of Axl Receptor Tyrosine Kinase Inhibitors: Suggestion of Combination Therapy 1

doi: 10.1016/j.neo.2014.03.009

Figure Lengend Snippet: Inhibition of the Axl RTK leads to up-regulation of HER3 phosphorylation. (A) Inhibition of Axl phosphorylation was determined 1 hour posttreatment with BMS777607 by p-Tyr–Axl ELISA in NIH/3T3-Axl cells. IC50 values were calculated by four-parameter log curve fit. (B) Axl inhibition led to an up-regulation of HER3 phosphorylation as determined by the Human Phospho-RTK Array Kit. MDA-MB231 cells were incubated with 1 μM BMS777607 for 24 hours or Axl-specific siRNA for 48 hours. The capture antibodies have been spotted in duplicates. The coordinates in the membrane for EGFR, HER2, Met, HER3, HER4, InR, IGF1R, and Axl are highlighted by rectangles. The corresponding antibody names are labeled at the bottom of the three displayed membranes from left to the right. (C) Validation of HER3 phosphorylation by HER3 immunoprecipitation with a HER3-specific antibody (Millipore, No. 05-390) and the Western blot for p-Tyr. The site-specific pHER3 Y1289 antibody displayed the same up-regulation of HER3 phosphorylation in Western blot experiments after treatment of MDA-MB231 cells with Axl-specific siRNA for 48 hours. Tubulin served as loading control. (D) Validation of HER3 phosphorylation by Western blot analysis of MDA-MB231 cells treated with Axl-specific siRNA for 48 hours or with 1 or 10 μM BMS777607 for 24 hours. The depletion of Axl kinase by siRNA was confirmed by anti-Axl Western blot analysis. Independent of the conditions, an increase of pHER3 Y1289 levels was evident in contrast to control treatments. Tubulin served as loading control. (E) Met-specific knockdown displayed no effect on pHER3 Y1289 levels compared to control siRNA treatment. The phosphorylation of HER3 Y1289 was induced by 10 μM BMS777607 treatment for 24 hours independent of Met expression. The depletion of Met kinase from the cells was confirmed by Western blot analysis performed on cell lysates harvested 48 hours after Met-specific siRNA transfection. Tubulin served as loading control.

Article Snippet: The Pan AKT-specific ELISA kit (R&D Systems GmbH, Wiesbaden-Nordenstadt, Germany, No. DYC887B-5) was used for quantification of phospho-AKT S473 according to the manufacturer's protocol with the following modifications: After the incubation with a biotinylated phospho-AKT (S473) panspecific detection antibody (1:180), we used an alkaline phosphatase–conjugated streptavidin SA110 (Millipore, Schwalbach, Germany) (1:4000) at room temperature.

Techniques: Inhibition, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Incubation, Membrane, Labeling, Biomarker Discovery, Immunoprecipitation, Western Blot, Control, Knockdown, Expressing, Transfection

Journal: Neoplasia (New York, N.Y.)

Article Title: Activation of HER3 Interferes with Antitumor Effects of Axl Receptor Tyrosine Kinase Inhibitors: Suggestion of Combination Therapy 1

doi: 10.1016/j.neo.2014.03.009

Figure Lengend Snippet: Inhibition of the Axl RTK activates HER3 transcription and phosphorylation. HER3 correlates with consumption of NRG1. (A) Quantification of HER3 mRNA induction in MDA-MB231 cells. Cells were treated with 10 μM BMS77607 for 1 hour up to 48 hours. A three- to six-fold increase of HER3 mRNA was evident between 16 to 48 hours posttreatment. Mean values and SEM are shown (n = 3). (B) Quantification of NRG1 mRNA induction with primer set 1 detecting all isoforms of NRG1 except isoforms No. 2 and No. 14. Cells were treated with 10 μM BMS777607 for 1 hour up to 48 hours. No significant increase of NRG1 mRNA levels was detected. Mean values and SEM are shown (n = 3). (C) Quantification of NRG1 mRNA induction with primer set 2 detecting isoforms No. 2, No. 15, and No. 17 of NRG1. Cells were treated with 10 μM BMS777607 for 1 hour up to 48 hours. No significant increase of NRG1 mRNA levels was detected. Mean values and SEM are shown (n = 3). (D) Quantification of NRG1 protein levels in conditioned medium was assayed using NRG1-ELISA. Cells were treated with 10 μM BMS777607 for 1 to 48 hours. A time-dependent consumption of NRG1 was measured. Mean values and SEM are shown (n = 3). (E) Western blot analysis of MDA-MB231 cells. Cells were treated with 10 μM BMS777607 for 1 to 48 hours. A significant increase of the pHER3 Y1289 levels was evident in MDA-MB231 after 6 hours of treatment. The up-regulation of HER3 protein expression was evident 16 hours posttreatment. BMS777607 treatment continuously suppressed the phosphorylation of AKT S473. Tubulin served as loading control. The diagrams show the densitometric analysis of Western blots for pHER3 Y1289 and HER3. Mean values and SEM are shown (n = 3).

Article Snippet: The Pan AKT-specific ELISA kit (R&D Systems GmbH, Wiesbaden-Nordenstadt, Germany, No. DYC887B-5) was used for quantification of phospho-AKT S473 according to the manufacturer's protocol with the following modifications: After the incubation with a biotinylated phospho-AKT (S473) panspecific detection antibody (1:180), we used an alkaline phosphatase–conjugated streptavidin SA110 (Millipore, Schwalbach, Germany) (1:4000) at room temperature.

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

Journal: Shock (Augusta, Ga.)

Article Title: Restorative mechanisms regulating protein balance in skeletal muscle during recovery from sepsis

doi: 10.1097/SHK.0000000000000762

Figure Lengend Snippet: A-F: Representative Western blots and quantitation are presented for phosphorylated and/or total Akt, PRAS40, TSC2, AMPK, and REDD1. The final panel is a representative tubulin blot demonstrating equal protein loading. Loading controls (tubulin or actin) were run for all proteins examined, but only representative data are shown. Bar graphs: were quantified for phosphorylation of each protein normalized to the total amount of the respective protein, except for REDD1 where total protein was normalized to tubulin. Control values were set to 100 arbitrary units (AU). Values are mean ± SEM. *P < 0.05 sepsis-recovery compared to pair-fed controls values; n = 17 and 10, respectively.

Article Snippet: Antibodies included (Cell Signaling, Beverly, MA, unless otherwise noted): S6K1, S6K1 (Thr389), rpS6, rpS6 (Ser240/244 and Ser230/235), 4E-BP1 (Bethyl Laboratories, Montgomery, TX), 4E-BP1 (Ser65), eukaryotic elongation factor (eEF)2, eEF2 (Thr56), eEF2 kinase, eEF2K (Ser366; Dr. Chris Proud), ERK (extracellular signal-regulated kinses), ERK1/2 (Thr202/Tyr204), RSK (90 kDa ribosomal S6 kinase), RSK1/2 (Ser380), REDD1 (regulated in development and DNA damage responses; ProteinTech, Chicago, IL), Akt, Akt (Thr308 and Ser473), PRAS40 (proline-rich Akt substrate of 40 kDa), PRAS40 (Thr246), eIF4B, eIF4B (Ser422), AMP-activated protein kinase-α (AMPK), AMPKα (Thr172), tuberous sclerosis complex 2 (TSC2), TSC2 (Thr1462), Unc-51 like autophagy activating kinase 1 (ULK1), ULK1 (Ser757), p62 (aka SQSTM1), light-chain 3B (LC3B)-I and –II, and MyoD and myogenin.

Techniques: Western Blot, Quantitation Assay