Anti perk1 2

General RSK1 (C-21; 1:4000), pRSK1/2 (T359, S381; 1:2000), pcMyc (T58, S62; 1:1000) and GAPDH (FL-335; 1:1000) antibodies (Abs) were obtained from Santa Cruz Biotechnology (CA, USA). Anti-CyclinD1 (92G2, 1:1000) and PARP (46D11, 1:1000) Abs were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-pERK1/2 (1:20000) and gERK1/2 (1:20000) Abs were obtained from Sigma (Rehovot, Israel). Anti-Sprouty2 (aminoterminal, 1:1000) Ab was obtained from Abcam (Cambridge, UK). Secondary fluorescent Ab conjugates were obtained from Jackson ImmunoResearch (West Grove, PA). Secondary Abs conjugated to horseradish peroxidase (HRP) were obtained from Nichirei Biosciences (Japan).

We used the following reagents: anti-p-p38 (Cell Signaling); anti-p-HSP27 (Enzo Life Sciences); anti-p38, anti-HSP27, anti-α-tubulin and anti-β-actin (Santa Cruz Biotechnology); anti-MKK3 and anti-MKK6 (Proteintech Europe); anti-p44/42 MAPK (ERK1/2) (Cell Signaling); anti-p-ERK1/2 (Sigma-Aldrich); anti-HERC1 (Bvg6, 411, 363, 410)^{25 (link)}; anti-C-RAF and anti-clathrin heavy chain (BD Biosciences); anti-A-RAF(A-5) and anti-B-RAF (F-7) (Santa Cruz Biotechnology); ubiquitylated proteins (clone FK2) (Biomol); anti-c-myc (clone 9E10) (Roche); anti-Flag M2 (Sigma-Aldrich); DAPI (Sigma-Aldrich); phalloidin-Alexa 647 (BioProbes); conjugated secondary antibodies to Alexa-Fluor 488 and horseradish peroxidase-conjugated secondary antibodies (Invitrogen); Immobilon-P PVDF transfer membrane (Millipore Corporation); SB203580 (Selleckchem); U0126 (Calbiochem); LY3009120 (Selleckchem); Sorafenib (Santa Cruz Biotech.); MG132 (Merck Millipore); anti-Flag M2 affinity gel (Sigma-Aldrich); Luminescent β-galactosidase detection Kit II (Clontech Laboratories); and Luciferase assay system (Promega).

Total cell lysate from 2D monolayer culture was prepared by extracting cells with modified RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS, 2 mM EDTA) supplemented with 50 mM NaF, 1 mM sodium pervanadate, and protease inhibitors. Total cell lysate from 3D culture was prepared by incubating cells at 4 °C in the modified RIPA buffer supplemented with 0.5% SDS and protease/phosphatase inhibitor cocktail (Pierce). The extracts were clarified by centrifugation at 13,000 rpm for 30 min and the supernatant was used for immunoblotting. Sources of different antibodies were: anti-Pfn1 (Abcam), anti-phospho-FAK (Y397) (Invitrogen), anti-Smad3 (Biorad), anti-ERK1/2, anti-phospho-ERK1/2 and anti-phospho-Smad3 (S423/S425) (Cell Signalling), anti-Pfn2 and anti-FAK (Santa Cruz), and anti-GAPDH and anti-tubulin (Sigma-Aldrich) Immunoblotting concentrations for different antibodies were: 1:3000 for anti-Pfn1, anti-GAPDH and anti-tubulin; 1:500 for anti-Pfn2, anti-ERK1/2, anti-pERK1/2 and anti-FAK; and 1:1000 for anti-Smad3, anti-pSmad3 and anti-pFAK.

Four adult flies of the corresponding feeding condition were lysed in 50 μl of EBR buffer [2 mM of Cl₂Ca, 129 mM of NaCl, 4 mM of KCl, and 35 mM of Tris (pH 6.85)] supplemented with phosphatase/protease inhibitor cocktail (Sigma #P2714), NaF (50 mM), and sodium orthovanadate (1 mM). A 12 mg protein/lane and a 24 mg protein/lane were used for pERK and pAKT immunoblots, respectively. Tissue samples were run on 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene difluoride (PVDF) (Thermo Scientific). Primary antibodies used were as follows: anti-pAKT^Ser505 1:1,000 (Cell Signaling #4054), anti-AKT 1:1,000 (Cell Signaling #9272), anti-pERK1/2 1:1,000 (Sigma #M8159), and anti-ERK1/2 1:15,000 (Sigma #M5670). Horseradish peroxidase (HRP)-conjugated secondary antibody goat anti-mouse 1:1,000 (Sigma #A5278), goat anti-rabbit 1:5,000 (Sigma #A0545), and Super Signal West Pico (Thermo Scientific) were used for signal detection in an ImageQuant RT ECL (GE Healthcare Life Sciences) device. Activation was quantified by ImageJ 1.47v as phospho-protein normalized to non-phospho-protein (pERK/ERK and pAKT/AKT).

Fibroblast cultures were homogenized with a sonicator in lysis buffer containing 20 mM Tris HCl, 10 mM potassium acetate (AcK), 1 mM dithiothreitol (DTT), 1 mM EDTA, 1 mM PMSF, 1 mM benzamidine, leupeptin, aprotinin, pepstatin 5 µg/ml each, 0.25% NP-40, pH 7.4, and then centrifuged at 12.000 g for 30 min at 4°C. For p-ERK and total ERK (microtubule-associated protein1 light chain 3) detection, 10 mM NaF, 2 mM sodium molibdate, 10 mM β-glicerophosphate, and 0.2 mM ortovanadate, were added to the lysis buffer. The supernatant was used for protein determination by the BCA protein assay kit and for electrophoretical separation. Samples (20–30 µg protein) were added to SDS sample loading buffer, electrophoresed in 10–15% SDS-polyacrylamide gels and then electroblotted to 0.45 µm nitrocellulose membranes, as described previously [19] (link), [20] (link).
The antibodies used in the study were the following: the chaperone mouse anti-HSP-70 (1/750) was from Santa Cruz (Heidelberg, Germany). Mouse monoclonal anti-huntingtin mAB 2166 (Chemicon, mAB 2166). Mouse monoclonal anti-ERK1/2 (SIGMA-Aldrich, M5670), Anti-P-ERK1/2 (SIGMA-Aldrich, M8159). Monoclonal anti- β-actin antibody diluted 1/5000 (SIGMA-Aldrich, A5441) diluted 1/10000 were used as a control of charge after inactivation of nitrocellulose membrane with sodium azide.

Immunohistochemistry was performed on paraformaldehyde-fixed floating coronal sections (20 μm, Bregma: 1.6 ± 0.6) from perfused rat brain. Sections were blocked (10% goat serum, 1% BSA, 0.1% Triton-X in 0.05M PBS) and then incubated for 60 h at 4°C with primary antibodies in buffer (2% goat serum, 0.01% Triton-X in 0.05M PBS) as previously described (Lee et al., 2004 (link); Hasbi et al., 2009 (link)). The primary antibodies used were: anti-phospho-Thr75-DARPP-32 (Cell signaling; 1:400), anti-phospho-Thr34-DARPP-32 (Cell Signaling; 1:400), and anti-total DARPP-32 (Cell Signaling; 1:400), anti-pERK1/2 (Sigma, 1:400), anti-D1R (Sigma, 1:300), anti-ENK (Millipore, mouse, 1:200), and anti-ΔfosB (Cell signaling, 14695S). After three washes with 0.05M PBS the samples were incubated with the appropriate secondary antibody in buffer for 2h at room temperature. The secondary antibodies conjugated to fluorophores (Alexa Fluor 488; Alexa Fluor 350, Alexa Fluor 568) were all purchased from Molecular Probes and used at 1:500. After three washes, the slides were mounted by using a mounting solution (Dako), and the images were acquired by using a confocal Fluoview Olympus microscope (FV 1000) with 40 × or 60 × /1.2 NA objective. Lower magnification images (40×) were used for cell counting. All images were acquired in sequential mode to minimize any bleed-through.

Anti-p-ERK1/2 (Sigma-Aldrich); anti-HERC1 (Bvg6 and 410) [46 (link)]; anti-Clathrin heavy chain and anti-C-RAF (BD Biosciences); anti-B-RAF (F-7) and anti-A-RAF(A-5) (Santa Cruz Biotechnology); anti-p44/42 MAPK (ERK1/2) and anti-His-Tag (27E8) (Cell Signaling); anti-GFP (Abcam); anti-α-Tubulin (Calbiochem); anti-ubiquitinylated proteins (clone FK2) (Biomol); horseradish peroxidase-conjugated secondary antibodies, and Alexa-Fluor 488 and 555 conjugated secondary antibodies (Invitrogen); anti-chicken IgY-peroxidases (Sigma-Aldrich) protein A-Sepharose and protein G-Sepharose (GE Healthcare); Phalloidin-Alexa 647 (BioProbes); DAPI (Sigma-Aldrich); Immobilon-P PVDF transfer membrane (Millipore Corp.); GFP-TrapA (Chromotek); U0126 (Calbiochem); Sorafenib (Santa Cruz Biotech.); LY3009120 (Selleckchem); MG132 (Merck Millipore); Ni-NTA Agarose (Qiagen); Recombinant Human His₆-Ubiquitin-activating Enzyme/UBE1 (Boston Biochem); EGF (PeproTech); Ubiquitin (human- recombinant) (Enzo).