Erk1 2 4695

Retinal tissue was harvested 24 h after IRI to investigate protein expression. Total protein from 75% of the retina was extracted and processed for Western blotting, as previously described [56 (link),57 (link)]. The membranes were blocked with 5% skim milk or bovine serum albumin in Tween20/PBS to minimize non-specific antibody binding and were subsequently incubated overnight with the following protein-specific antibodies, diluted as recommended by the manufacturer (iNOS#130246, VEGF#102643, ICAM-1#100450, caspase 3 #110543, cleaved caspase #86952, all Genetex, Irvine, CA, USA; ß-actin #3700, p-p38 #4511, p38 8690, p-ERK1/2 #9101, ERK1/2 #4695, all Cell Signaling Technology, Danvers, MA, USA). After incubation with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (GE Healthcare, Freiburg, Germany), the proteins were visualized with an enhanced chemiluminescence Western blotting detection reagent (Western Lightning plus enhanced chemiluminescence, #NEL104001EA, PerkinElmer, Waltham, MA, USA), according to the manufacturer’s instructions to visualize the proteins. Relative changes in protein expression in retinas exposed to IRI in the four treatment groups were calculated based on the untreated retinas of each respective animal. The chemiluminescence imaging system Fusion Fx^® (Vilber, Collegién, France) was employed for recordings and densitometric analysis.

Cells were gently washed two times in PBS and then lysed in sample buffer 2X. The extracts were sonicated (50 W, 30 s), incubated on ice for 15 min, and boiled for 5 min. The samples were then subjected to 10% SDS‐PAGE. Immunoblots were probed with the following antibodies: pERK1/2 (4370S;1:1000), ERK1/2 (4695;1:1000), P38 (9212S; 1:1000), pP38 (9216S; 1:1000), JNK (9258;1:1000), and pJNK (9251S;1:1000) antibodies were obtained from Cell Signaling. GAPDH (MAB374;1:1000), Tubulin (05–829;1:1000), RAS, and RAS‐GTP (RAS activation kit) antibodies were obtained from Millipore. FBXO42 (ab81638;1:500) was obtained from Abcam.
Blots were developed with HRP‐conjugated anti‐mouse or anti‐rabbit Abs, using SuperSignal West Pico Chemiluminescent Substrate or SuperSignal West Femto Chemiluminescent Substrate from Thermo Scientific. pERK trametinib‐treated Western blots were developed using SuperSignal West Femto Chemiluminescent. Pictures of the blots were taken using Bio‐Rad ChemiDoc MP System. Quantification was done using Image Lab (Bio‐Rad).

Western blots were performed as previously described [9 (link), 10 (link)] using the primary antibodies against MKL1 (ab14984) from Abcam, ERα (sc-543), and p-ERK (sc-7383) from Santa Cruz Biotechnology, ERK 1/2 (4695) from Cell signaling technology, and p-mTOR (5536) from Cell Signaling Technology.

Cells were lysed with radioimmunoprecipitation assay buffer (RIPA Bufer, Thermo Scientific) containing protease inhibitors (Complete, Mini; Protease Inhibitor Cocktail Tablets, Roche) and phosphatase inhibitors (PhoStop, Phospatase Inhibitor Cocktail Tablets, Roche) and centrifuged at 13000×g, at 4ºC, for 20 minutes. The protein content of the supernatants was determined with BCA assay system (Pierce). Lysate aliquots (50 μg) were resolved by 8%, 10% or 12% (depending on the protein molecular weight) SDS-PAGE and transferred onto nitrocellulose membranes. After blocking with 5% skimmed milk in PBS containing 0.2% Tween 20 at room temperature for 1 hour, membranes were incubated overnight at 4ºC with the appropriate primary antibody (CAV1 #610059 from BD, FoxO1 #2880, ERK1/2 #4695, phospho-ERK1/2 #4376 from Cell Signaling Technology; CAV3 #sc5310 and Myogenin #sc-12732 from Santa Cruz; MyHC MF 20 from Developmental Studies Hybridoma Bank). Blots were then incubated at room temperature for 1 hour with a horseradish peroxidase–conjugated secondary antibody and the peroxidase activity was detected by enhanced chemiluminescence (Pierce) following the instructions of the manufacturer. Immunodetection of β-actin (#ab49900) from Abcam was used as a loading reference.

MCF-7, a human breast carcinoma cell line derived from the pleural effusion metastasis, weas purchased from ATCC (USA). Dox was purchased from Farmitalia (Italy). MCF-7 resistant cells (MCF-7R) were obtained from MCF-7 parental cells by increased Dox treatment. DMEM/F-12, trypsin and Lipofectamine RNAiMAX were from Invitrogen (France). Bovine fetal serum was from Dutscher (France). LRP-1 polyclonal antibody ‘ab92544’ was from Abcam (France). Caspase-7 ‘#9492’, ERK 1/2 ‘#4695’, Phospho-ERK 1/2 ‘#4370’, LAMP-1 ‘#9091’ and P-gp ‘#13342’ antibodies were from Cell Signaling Technology (France). LRP-1 siRNA kit ‘sc-40101’ was purchased from Santa Cruz Biotechnology (USA). Kit ECL was from Amersham (Germany). U0216, a elective inhibitor of MEK-1 and MEK-2, was from Cell Signaling Technology (France). UptiBlue and BCA kit ‘23225’ were from Uptima (Thermofisher, France). CaspACE assay kit ‘G8091’ was from Promega (France). β-actin antibody ‘A5316’, invertase enzyme ‘I9274’ and all other reagents were from Sigma (USA). Histidine-tagged RAP was purified as previously described [41 (link)] and used as an antagonist of LRP-1-dependent endocytosis [41 (link), 42 (link)].