Phospho p44 42 mapk erk1 2

Antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), with the exception of anti-p44/42 MAP Kinase antibody and the following phospho-specific antibodies which were obtained from Cell Signaling Technologies (Danvers, MA, USA): Phospho Syk (Tyr525/526); Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204); Phospho-SAPK/JNK (Thr183/Tyr185); Phospho-p38 MAP Kinase (Thr180/Tyr182); Phospho-Src Family (Tyr416); Phospho-NFkB (Ser536); Phospho-Gab2 (Tyr452); Phospho-PLCγ2 (Tyr1217); Phospho-Akt (Ser473). The anti-human C12orf4 antibody and all reagents were obtained from Sigma-Aldrich (St Louis, MO, USA). Antiphosphotyrosine mAb 4G10 was purchased from Upstate Biotechnology (Millipore, MA, USA). Alexa 488 conjugated anti-mouse IgG and Alexa 594 conjugated anti-rabbit IgG antibodies were purchased from Jackson ImmunoResearch laboratories (West Grove, PA, USA).

A427 cells stably transfected with plasmids containing either murine full-length Lrp1b or empty vector (EV) control were serum-starved overnight and subsequently stimulated with 10% fetal calf serum. Total cell extracts from these cells were analyzed using the following antibodies: Phospho-Akt (Cell Signaling Technology #9271); Phospho-p44/42 MAPK (Erk1/2; Cell Signaling Technology #9101); p21^CIP1 (BD Pharmingen #556430); Phospho-Src (Tyr416; Cell Signaling Technology #2101); Phospho-Src (Tyr527; Cell Signaling Technology #2105); Phospho-Stat3 (Ser727; Cell Signaling Technology #9134); Phospho-Stat3 (Tyr705; Cell Signaling Technology Antibody #9131); GAPDH (Abcam #ab37187).

CRC cells (SW480 and SW620) were transfected with NNT-AS1 siRNAs or Control siRNA in 6-well plates. Seventy-two hours after transfection, the cells were washed twice with cold PBS, and 30 μL of RIPA (Solarbio, Shanghai, China) containing a protease inhibitor cocktail (Invitrogen, Carlsbad, CA, USA), a phosphatase inhibitor cocktail (Invitrogen, Carlsbad, CA, USA) and 2 mM ethylenediaminetetraacetic acid (EDTA) at pH 8.0 was added to each well. Following this, the cells were collected into a cold tube. Cell lysates were centrifuged at 12,000 g for 20 min at 4°C. Subsequently, the proteins in the lysates were separated on a 10% polyacrylamide gel and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 10% non-fat milk for 4 h at room temperature and then incubated with primary antibodies for 1 h at 4°C. Antibodies against the following proteins were used: E-cadherin (24E10), vimentin (D21H3), p44/42 MAPK (Erk1/2), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E), GAPDH (D16H11) (from Cell Signaling Technology, Danvers, MA) and Ras [EP1125Y] (from Abcam, Cambridge, UK), followed by second antibody (zhongshanjinqiao, China). Protein bands were visualized using Super Enhanced Chemiluminescence Detection regents (Applygen Technologies, Beijing, China).

Zebrafish were euthanized in tricaine anesthetic, fixed in 4% paraformaldehyde at 4°C for 2 days, and decalcified with 0.25 M EDTA, pH 8.0, for at least 24 hr. Paraffin sectioning followed by hematoxylin and eosin (H&E) staining or immunohistochemistry (IHC) was performed at the Dana-Farber/Harvard Cancer Center Research Pathology Core. Primary antibodies included Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204, Cell Signaling #4370; 1:150), Phospho-AKT (Ser473, Cell Signaling #4060), Phospho-S6 ribosomal protein (Ser240/244, Cell Signaling #4838), PCNA (PC10, EMD Millipore; 1:100), cleaved Caspase-3 (Cell Signaling #9664; 1:250), TH (Pel-Freez # P40101, 1:500) and HuC/D (Invitrogen #A-21271, 1:200). Antibody binding was detected with a diaminobenzidine-peroxidase (DAB) visualization system (EnVision+, Dako, Carpinteria, CA). Mayer’s hematoxylin was used for counterstaining.

After treatment of cells with the indicated cytokines or inhibitors for the indicated times, cells were washed twice with ice-cold PBS and scraped from the plastic plate with a cell lifter (Costar), and then whole-cell lysates were isolated in RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 25 mm Tris-HCl, pH 7.6, 150 mm NaCl) supplemented with protease inhibitor mixture and PhosSTOP (Roche Applied Science). Equivalent amounts of protein (20–40 μg) were subjected to 10% SDS-PAGE, and immunoblotting was performed using antibodies specific for phospho-Akt (Ser-473), Akt, phospho-p38 MAPK (Thr-180/Tyr-182), p38, phospho-p44/42 MAPK (ERK1/2) (Thr-202/Tyr-204), ERK1/2, JNK (Cell Signaling), and phospho-JNK (Thr-183/Tyr-185) (BD Biosciences). For RhoA activation assay, cell lysates were incubated with Rhotekin-RBD protein beads and blotted with anti-RhoA mAb (Cytoskeleton).

Immunohistochemical staining of skin sections with specific antibodies [phospho-p44/42 MAPK (ERK1/2) (#4370, Cell Signaling Technology; 1:400), phospho-mTOR (#2976, Cell Signaling Technology; 1:100) and Ki-67 (Thermo Fisher Scientific; prediluted)] was performed on a Ventana Discovery XT automated IHC research slide staining system (Roche, Tucson, USA). For antigen unmasking, deparaffinized slides were pretreated with Cell Conditioning Solution (Ki-67) or citrate buffer (10 mM Sodium Citrate, 0.05% Tween 20, pH 6.0) (phospho-ERK1/2 and phospho-mTOR). Ki-67 proliferation index was determined as described previously (Almeida et al., 2012 (link)).