M9692

Immunostaining was performed essentially as previously described [120 (link)] using the following primary antibodies: anti-collagen II (DSHB II-II6B3-c, 1:200), rabbit anti-RFP (Abcam; ab62341, 1:200), chicken anti-GFP (Invitrogen; A10262, 1:300), mouse anti-p63 BC4A4 (Zytomend, 1:200), and mouse anti-MAP Kinase, activated (Sigma, M9692, 1:50). For pAkt staining, cryosections of EAF-fixed embryos were washed with PBS-TritonX, followed by an antigen retrieval in 10 mM Tris, 1 mM EDTA, pH 9.0 for 5 min at 98°C, blocking in 5% sheep serum and antibody incubation with rabbit anti-pAkt (S473) (Cell signaling #4060, 1:50) (modified after [121 (link)]). Secondary antibodies were anti-rabbitAlexa555, anti-mouseAlexa488, anti-mouseAlexa647, and anti-chickenAlexa488 (all Invitrogen, 1:200). Sections were mounted either directly in Mowiol containing DAPI or stained with a 1:1000 dilution of CellMask Deep Red Plasma membrane Stain (Molecular Probes) in PBS for 10 min and washed with PBS several times prior to mounting. Images were taken using a Zeiss Confocal (LSM710 META).

To assess for the presence of endogenous and recombinant expression of ERK1, mouse eggs were collected in SDS buffer, heated for 5 min at 100°C and proteins were separated by SDS-PAGE (Nomikos et al., 2011 (link)). Immunoblotting was then performed as described previously (Verlhac et al., 1996 (link)). Following transfer onto polyvinylidene difluoride membrane (Immobilon-P; Millipore) using a semi-dry transfer system (Trans-Blot SD; Bio-Rad) in buffer (48 mM Tris-HCl, 39 mM glycine, 0.0375% SDS) at 22 V for 4 h and blocking overnight in 5% skimmed (low-fat) milk in TBS (10 mM Tris-HCl, pH 7.5, 140 mM NaCl) containing 0.1% Tween-20 (TBS/Tween), the membrane was incubated for 1 h with the appropriate primary antibody. ERK1 was detected using monoclonal antibody against diphosphorylated ERK1/2 (1∶1000, M9692 Sigma) and ERK1 (1∶500, G-8; sc-271269, Santa Cruz Biotechnology) antibodies. Detection of horseradish-peroxidase-coupled secondary antibody was achieved using enhanced chemiluminescence detection (ECL, Amersham Biosciences). All experiments were repeated at least three times.

Primary antibodies were sourced as follows: p-MYPT1 Ser-507, antibody 3040; p-MYPT1 Ser-668, antibody 3048; p-MYPT1 Thr-853, antibody 4563; p-MYPT1 Thr-696, antibody 5163; MYPT1, antibody 2634; p-RSK T359/S363, antibody 9344; p-RSK Thr-359, antibody 8753; p-RSK Ser-380, antibody 9341; pan-RSK, antibody 9355; RSK1, antibody 8408; RSK2, antibody 5528; p-AKT Ser-473, antibody 4060; AKT, antibody 9272; S6K, antibody 9202; p-S6 235/236, antibody 4858; S6, antibody 2317; p-p38, antibody 9211; ERK, antibody 9102 (CST); FLAG M5 and p-ERK, antibody M9692 (Sigma); HA.11 (Covance), ROCK1 antibody A300-455 and A300-457 (Bethyl); MLC, antibody ab92721; and p-MLC S20, antibody ab2480 (Abcam). 680LT and 800CW IRDye-labeled secondary antibodies were from Li-COR or Fisher. Membranes for phospho-MYPT1, MYPT1, and myosin antibodies were first blocked with 5% (w/v) BSA in TBS (10 mm Tris, pH 7.4, and 150 mm NaCl), followed by primary antibody incubation in a 1:2 mixture of Odyssey blocking buffer and TBS. All others were blocked with 5% nonfat dry milk in TBS. After incubation with primary and secondary antibodies, the membranes were washed with TBS-T (TBS with 0.1% Tween 20). Western blots were imaged on a Li-COR Odyssey Imager. Marker lanes visible in the 680 channel scans were overlaid onto the 800 channel scans.