Horseradish peroxidase hrp conjugated secondary antibody

Aortas and SMA vessels were dissected, homogenized, and lysed. Proteins (80 μg) were separated on 10% SDS-PAGE. Blots were probed with anticyclooxygenase- (COX-) 1 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-COX-2 (BD Biosciences, San Jose, CA), and anti-NF-κB p65 (Abcam, Paris, France) antibodies. A monoclonal mouse β-actin antibody (Sigma-Aldrich) was used at 1/5000 dilution for visualization of protein gel loading. The membranes were then washed at least three times in Tris buffer solution containing 0.05% Tween and incubated for 1 hour per wash at room temperature, with the appropriate horseradish peroxidase- (HRP-) conjugated secondary antibody (Amersham). The protein antibody complexes were detected by ECL plus (Amersham) according to the protocol of the manufacturer as previously done [29 (link), 30 (link)].

Total corneal protein was used for the detection of Western blot, which was extracted by lysis in radio immunoprecipitation assay (RIPA) buffer containing a proteinase inhibitor cocktail. Then, the extracted protein samples (10‐20 µg total proteins) were run on 10% or 12% SDS‐PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). The protein‐loaded membranes were incubated with primary antibodies (showed in Table 1) overnight at 4°C and then followed by the incubation of horseradish peroxidase (HRP)‐conjugated secondary antibody (Amersham Biosciences) for 1 hour at room temperature. The target proteins were developed by the application of enzyme‐linked chemiluminescence (ECL) kit (Pierce), and ImageJ software was used for the quantification of immunoreactive bands.

Total protein was lysed from TKE2 cells in RIPA buffer. Samples were run on 12% SDS-PAGE gels for 2 h at 110 V and then transferred to a PVDF membrane (Millipore, Billerica, MA). The blots were blocked in 5% non-fat dried milk in TBST for 1 h at room temperature, and incubated overnight with primary antibodies against p-Akt (1:2000), total Akt (1:2000, Epitomics), p-ERK1/2 (1:1000, abcam), total ERK1/2 (1:1000, abcam), p-p38 (1:300, abcam), total p38 (1:1000, abcam), p-mTOR (1:2000, abcam) and total mTOR (1:1000, abcam) at 4°C. After three washes with TBST, the blots were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Amersham Biosciences, Piscataway, NJ) and visualized via enzyme-linked chemiluminescence using the ECL kit (Chemicon, Temecula, CA). The blots were analyzed by Image J software with total Akt, ERK1/2, p38 or mTOR as control.

Protein from combined cerebral cortex and hippocampus was extracted with RIPA Buffer (Sigma Aldrich) containing a cocktail of protease inhibitors (Roche). Protein extract was then placed at 4 °C for 30 min, centrifuged at 13,000 rpm for 20 min and supernatant stored at −80 °C.
Denatured proteins samples (15 μg) from each time-point were electrophoresed into 10 % SDS-PAGE gels (BioRad), transferred to PVDF membranes (BioRad) and incubated in primary antibodies overnight (Table 1). A corresponding anti-rabbit or anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (1:7000; Amersham) was used, as described previously [18 ]. GAPDH (1:7000, Millipore) was used as a loading control and band intensity was measured as the integrated intensity using ImageJ software (v1.4; NIH), and normalized with respect to the loading controls. Three experimental repeats were carried out.Table 1

List of qPCR primers

Gene name	Forward primer	Reverse primer	Accession number
C9orf72 isoform 1	5’- CCCACCATCTCCTGCTGTTG-3’	5’-GTAAGCAAAGGTAGCCGCCA-3’	NM_001081343.1
C9orf72 isoform 2	5’- TGGAAGATCAGGGTCAGAGT-3’	5’- GCAAGCAGCTCCATTACAGG-3’	XM_006538294.1
C9orf72 isoform 3	5’-CTTTCCTTGCACAGTTCCTCC-3’	5’- TCATCCTCGATGTACTTGATTAGTG-3’	XM_006538292.2

Primers used for qPCR analysis of the 3 C9orf72 isoforms including primer sequence (forward and reverse sequence respectively) and GenBank accession number

PM monolayers were lysed in 1% Triton X-100 buffer containing 50 mM Tris [tris(hydroxymethyl)aminomethane] (pH 8), 100 mM NaCl, 1 mM Na₃VO₄, 1 mM PMSF (phenylmethylsulfonyl fluoride), 50 mM NaF, and 1 μg·ml⁻¹ leupeptin. Lysates were separated on SDS 8% polyacrylamide gel electrophoresis (SDS-PAGE) gels. The proteins were transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH) overnight at 100 amps (A) and for 3 h at 200 A. Next, the membrane was blocked with 5% fat-free milk in TBST 0.1% for 1 h, washed 3 times, and then probed with antiphosphorylated ERK-1/2 (Cell Signaling) for 1.5 h. After that, the membranes were washed and incubated with a horseradish-peroxidase- (HRP-) conjugated secondary antibody (1 : 10,000; Amersham Pharmacia Biotech, Piscataway, NJ). Phosphorylated bands were visualized using the enhanced chemiluminescence system (ECL, Amersham, Arlington Heights, IL). The membranes were then stripped, blocked, and reprobed with anti-ERK-1/2 or anti-GAPDH for 1 h followed by an incubation with HRP-conjugated secondary antibody (1 : 15,000; Amersham Pharmacia Biotech). The bands were visualized using the ECL system.

The protein concentration was measured by the bicinchoninic acid (BCA) quantification assay (Pierce Biotechnology, Rockford, IL, USA). Equal amounts (20 μg) of whole protein extract were electrophoresed on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes using a semidry transfer cell (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were incubated overnight at 4°C with specific primary antibodies against α-synuclein (1:1000; Abcam, USA) and β-actin (1:1000; Abcam, USA) after being blocked with 5% nonfat dry milk to prevent the nonspecific binding of the primary antibody. Horseradish peroxidase (HRP) conjugated secondary antibody (1:10000; Amersham, Arlington Heights, IL) was used to incubate the blot for one hour at room temperature on a rocking platform. Besides, the blot was washed with 1x TBST three times. Finally, super signal chemiluminescence reagents (Thermo Fisher Scientific, Inc., Massachusetts, USA) were used to detect signal intensities.