Rabbit monoclonal anti erk1 2

Western blotting analysis was performed as previously described [62 (link)] using the following primary antibodies: rabbit polyclonal anti-TH (ab112, 1:200), rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; ab7260, 1:1500), rabbit polyclonal anti-c-FOS (ab7963, 1:500), rabbit polyclonal anti-IL-1β (ab9722, 1:500), mouse monoclonal anti-IL6 (ab9324, 1:500), and rabbit polyclonal anti-TNF-α (ab6671, 1:500) (all from Abcam, Cambridge, MA, USA); rabbit monoclonal anti-ERK1/2 (#4695, 1:1000), rabbit monoclonal anti-p-ERK1/2 (#4377, 1:500) (both from Cell Signaling Technology, Danvers, MA, USA); and rabbit polyclonal anti-Gsto2 (14562-1-AP, 1:1000) and rabbit polyclonal anti-PTGER3 (14357-1-AP, 1:500) (both from Proteintech, Rosemont, IL, USA). Rabbit monoclonal anti-GAPDH antibody (Abcam; ab181602, 1:3000) was used for the loading control. Protein band density was quantified using an Epson V330 Photo scanner (Seiko Epson, Nagano, Japan) and Quantity One software (Bio-Rad, Hercules, CA, USA).

For western blotting, the following antibodies were used: mouse monoclonal anti-MTBP (sc-137201, Santa Cruz), rabbit polyclonal anti-GAPDH (sc-27117, Santa Cruz), goat polyclonal anti-Lamin B (sc-6216, Santa Cruz), rabbit monoclonal anti-Elk-1 (# 9182, Cell Signaling), rabbit monoclonal anti-p-Elk-1 Ser383 (# 9181, Cell Signaling), rabbit monoclonal anti-Erk1/2 (# 4695, Cell Signaling), mouse monoclonal anti-p-Erk1/2 (# 9106, Cell Signaling), rabbit monoclonal anti-importin 7 (PAS5-25349, Thermo Scientific), mouse monoclonal anti-vinculin (10R-C105a, Fitzgerald, Acton, MA, USA). For co-immunoprecipitation studies; antibodies for mouse monoclonal anti-importin 7 (sc-271701, Santa Cruz), goat polyclonal anti-MTBP (sc-4717, Santa Cruz), rabbit monoclonal anti-p-Erk1/2 Thr202/Tyr204 (#4370, Cell Signaling), and mouse monoclonal anti-FLAG (M2, Sigma) were used. For immunofluorescence and IHC studies, antibodies for goat polyclonal anti-MTBP (N-13, sc-47174, Santa Cruz) and rabbit monoclonal anti-p-Erk1/2 Thr202/Tyr204 (#4370, Cell Signaling) were used.

Striatum proteins of the Sham, PD, NLID, and LID rats were extracted using a protein extraction kit (GPP1814; GenePool, Beijing, China). Rabbit polyclonal anti-TH (ab112; 1:200), rabbit polyclonal anti-c-FOS (ab7963; 1:500) (both from Abcam); rabbit monoclonal anti-ERK1/2 (#4695; 1:1000), and rabbit monoclonal anti-p-ERK1/2 (#4377; 1:500) (both from Cell Signaling Technology, Danvers, MA, USA) were used as the primary antibodies. Rabbit monoclonal anti-GAPDH antibody (ab181602; 1:3000; Abcam) was used for the loading control. Protein band density was quantified using the Quantity One software (version 4.6.2; Bio-Rad, Hercules, CA, USA).

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and 0.25% trypsin-EDTA, penicillin and streptomycin (PenStrep) were purchased from Invitrogen (Carlsbad, CA, USA). 5-(and-6)-chloromethyl-2,7-dichlorodihydrofluoresceindiacetate, acetyl ester (CM-H2DCFDA) was obtained from Molecular Probe (Invitrogen). LPS (Escherichia coli 0127:B8) was purchased from Sigma-Aldrich (St. Louis, MO). The following primary antibodies were used in this study: rabbit monoclonal anti-Nox4,rabbit polyclonal anti-TNFα, rabbit polyclonal anti-TGFβ1 (Abcam, Cambridge, MA, USA); rabbit polyclonal anti-Nox4 (Millipore, Billerica, MA, USA); rabbit polyclonal anti-NFĸB p65, rabbit polyclonal anti-PCNA, goat polyclonal anti-GAPDH, goat polyclonal anti-actin (Santa Cruz Biotechnology, CA, USA); rabbit monoclonal anti-ERK1/2, rabbit monoclonal anti-MyD88 (Cell Signaling Technology, MA, USA); mouse monoclonal Anti-ERK1/2 (pT202/pY204) (BD Biosciences, USA) and rabbit polyclonal anti-NFĸB p65(Ser536) (Bioss Antibodies, USA).

Cells grown and treated as indicated were collected and total protein was extracted. The following primary antibodies were used: rabbit monoclonal anti-α-actin (Abcam), monoclonal anti-COL1A1 (Millipore), rabbit monoclonal anti-phosphorylated SMAD2, rabbit monoclonal anti-phosphorylated SMAD3, rabbit monoclonal anti- SMAD2/3, rabbit monoclonal anti-ERK1/2, or rabbit monoclonal anti-phosphorylated ERK1/2, rabbit monoclonal anti-Akt, rabbit monoclonal anti-phosphorylated Akt, rabbit monoclonal anti-STAT3, or rabbit monoclonal anti-phosphorylated STAT3 (all from Cell Signaling Technology, Inc.). Horseradish peroxidase-conjugated goat-anti-rabbit antibody (Thermo Scientific) was used as a secondary antibody. The control for equal protein loading was assessed with an anti-β-actin antibody (Cell Signaling Technology, Inc.).

Antibodies used were: mouse monoclonal anti-Myc tag (9b11) (Cell Signaling #2276) used at 1:1000 for immunoblot analysis, 1:400 for immunostainings and 1:500 for immunoprecipitation, rat monoclonal anti-HA (3F10) (Roche #11867423001) used at 1:1000 for immunoblot analysis and 1:400 for immunostainings, rabbit anti-pS6K (Thr389) (Cell Signaling #9205) used at 1:1000 for immunoblot analysis, rabbit monoclonal anti-ERK1/2 (Cell Signaling #4695) used at 1:2000 for immunoblot analysis.

Ipconazole-treated 3-dpf larvae were homogenized with a plastic pestle in 1x RIPA buffer (IBS-BR004, iNtRON, Seongnam, Republic of Korea) containing 1x Xpert duo inhibitor cocktail solution (P3300, GenDEPOT, Baker, TX, USA) and centrifuged. The supernatant containing total protein was prepared as an SDS sample buffer. Samples were electrophoresed on 13% SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Merck, Darmstadt, Germany). Membranes were blocked at 23 °C with 5% skim milk/TBST or 5% BSA/TBST and incubated overnight at 4 °C with primary antibodies: rabbit monoclonal anti ERK1/2 (CST #4370, Cell Signaling Technology, Beverly, MA, USA) in 5% skim milk/TBST (1:1000); rabbit monoclonal phospho-ERK1/2 (137F5) (CST #4695, Cell Signaling Technology, MA, USA) in 5% BSA/TBST (1:2000); mouse monoclonal anti-β-actin (sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA) in 5% skim milk/TBST (1:2500). The secondary antibodies used were HRP-conjugated anti-rabbit (GenDEPOT, SA002) (1:2000) or HRP-conjugated goat anti-mouse (SA001, GenDEPOT) (1:20,000). Protein bands were imaged using a chemiluminescence substrate kit (W365100-012, GenDEPOT) and the ImageQuant LAS 500 imaging system (General Electric Healthcare, Chicago, IL, USA), and relative quantification was performed using ImageJ 1.53q (U. S. National Institutes of Health, Bethesda, MD, USA).