Mouse monoclonal anti gfp

Polyclonal rabbit anti-GFP and anti-Myc were generated as described (Mizutani et al, 2013 (link)). Mouse monoclonal glial fibrillary acidic protein (GFAP) antibody was from Chemicon (Temecula, CA, USA). Goat polyclonal anti-HSPA5, mouse monoclonal anti-GFP and anti-β-tubulin were from Santa Cruz Biotech. (Santa Cruz, CA, USA). Goat polyclonal anti-SIL1 was from abcam (Tokyo, Japan). Mouse monoclonal anti-Flag M2 and rabbit polyclonal anti-Flag were from Sigma (Tokyo, Japan). Rabbit polyclonal anti-active caspase3 and anti-Ki67 were from Cell Signaling Technology (Danvers, MA, USA) and Thermo Scientific Japan (Yokohama, Japan), respectively.

Cells were lysed with a buffer containing 50 mM Tris–HCl pH 8.0, 150 mM sodium chloride, 0.5% NP-40, 5 mM EDTA, 10 mM sodium fluoride, 1 mM sodium orthovanadate, and a protease inhibitor cocktail tablet (Roche Diagnostics Corporation, Indianapolis, IN, USA). Proteins were separated by SDS-PAGE and analyzed by western blotting with mouse monoclonal anti-tubulin (1∶1000) (12G10, Developmental Studies Hybridoma Bank, The University of Iowa, IA, USA), mouse monoclonal anti-actin (1∶1000), mouse monoclonal anti-GFP (1∶1000) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), rabbit polyclonal anti-AprA (1∶1000) [41] (link), and rabbit polyclonal anti-CfaD (1∶1000) [42] (link). Immunoblots were digitally scanned using a GS800 Calibrated Densitometer scanner and Quantity One software (Bio-Rad Laboratories Incorporated, Hercules, CA, USA). Identified bands were quantified with ImageJ/Fiji and levels were normalized to ß-actin levels. Results were pooled from four independent experiments, each with at least two technical replicates. Statistical significance was determined using a one-sample t-test (mean, 100; two-tailed). A p-value<0.05 was considered significant.

Protein extracts were prepared as described previously [23] (link). The blot was probed with mouse monoclonal β-actin (1∶1000, Santa Cruz, CA, USA), rabbit monoclonal anti-GATA-1 (1∶500, Cell Signaling, Danvers, MA, USA), rabbit monoclonal anti-GATA-2 (1∶500, Abcam, Cambridge, UK), rabbit polyclonal anti-postsynaptic density protein 95 (PSD-95) (1∶1000, Abcam, Cambridge, UK), rabbit polyclonal anti-GAP43 (1∶1000, Abcam, Cambridge, UK), mouse monoclonal anti-Tuj1 (1∶1000, Covance, Berkeley, CA, USA), rabbit polyclonal anti-lamin B1 (1∶1000, Abcam, Cambridge, UK), mouse monoclonal anti-GFP (1∶500, Santa Cruz, CA, USA) followed by treatment goat with anti-mouse or anti-rabbit IgG conjugated with peroxidase (1∶1000, Santa Cruz, CA, USA). Bands were visualized with an ECL detection kit (GenDEPOT, TX, USA).

At 4 weeks after surgical operation (postop), animals were humanely killed under deep anesthesia. Samples were extracted and fixed in 4% paraformaldehyde. After decalcification, the specimens were embedded in paraffin and sectioned into 4 μm thick sections. For morphological study, sections were stained with hematoxylin and eosin (HE) with an autostainer (ST5010, Leica, Germany). For immunohistochemistry analysis, sections were subjected to immunostaining with mouse monoclonal anti-GFP (1 : 100, Santa Cruz, USA), rabbit polyclonal anti-rat osteocalcin (OCN) (1 : 100, Santa Cruz, USA), and rabbit polyclonal anti-human OPN antibodies (1 : 100, Proteintech Group, USA). Then all samples were photographed.

For protein extraction and immunoblotting cells were lysed as described below (Mass spectrometry). 40 µl of the lysed supernatant was resuspended in 6x Laemmli loading buffer. Samples were resolved on 10% SDS-polyacrylamide gels and blotted onto nitrocellulose membranes. Membranes were blocked using 3% milk in TBST (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween 20) for 1 h at room temperature and stained with mouse monoclonal anti-GFP (1:2000, Santa Cruz Biotechnology, #sc-9996) in 3% milk in TBST at 4°C overnight, followed by three 5-min washes in TBST. Membranes were then stained with Alexa Fluor 680–conjugated goat anti-mouse antibody (1:20,000; Jackson Laboratory; #115-625-166) in 3% milk in TBST at room temperature, followed by three 5-min washes in TBST. Images were developed using a LI-COR processor.

The following reagents were used: monoclonal anti-KDEL (Abcam; ab176333), monoclonal anti-Tom20 (Santa Cruz Biotech., (F-10): Cat# sc-17764), monoclonal anti-PMP70 (Abcam; Cat# ab211533), monoclonal anti-STIM1 (BD Transduction Laboratories^TM; Cat# 610954), polyclonal anti-Orai (Proscience; Cat# 30-571), monoclonal anti-β-actin (Sigma-Aldrich; Cat# A5441), mouse monoclonal anti-GFP (Santa Cruz Biotech.: Cat# sc-9996), polyclonal anti-β-tubulin (Cell Signaling; Cat# 2146c), AlexaFluor secondary antibody fluorophore-conjugated (Thermo Fisher: Goat anti-Rabbit IgG AlexaFluor 405, Cat# A-31556; Goat anti-Mouse IgG AlexaFluor 405, Cat#A-31553; Donkey anti-Rabbit IgG AlexaFluor 647, Cat# A-32795), secondary horseradish peroxidase-conjugated antibodies (Santa Cruz Biotech.; Goat anti-Rabbit IgG-HRP, Cat#sc-2004; Goat anti-Mouse IgG-HRP, Cat#sc-2005), 1:5000 in TBST.