Anti rabbit hrp conjugated secondary antibody

Western blotting was performed on samples containing 60 wing imaginal discs. Tubulin was used as a loading control. Mouse anti-Tubulin primary antibody (Sigma, cat. no. T9026) was used at a 1:2,000 dilution with an anti-mouse-HRP conjugated secondary antibody (Sigma, cat. no. A2304) at 1:80,000. Rabbit anti-Dis3⁷ (link) was used at 1:1,500 with an anti-rabbit-HRP conjugated secondary antibody (Sigma, cat. no. 1949) at 1:80,000. Antibody binding was detected using Amersham ECL detection reagents (GE Healthcare, cat. no. RPN2209). Relative quantification of bands was performed with ImageJ software.

Cells were fixed and total protein extracts prepared by precipitation with trichloroacetic acid (TCA) as previously described53 (link). Proteins were resolved in 10% SDS-PAGE gels and transferred to Hybond-ECL membranes. An anti-phospho-polyclonal antibody produced by immunization of rabbits with a synthetic phospho-peptide corresponding to residues surrounding Ser546 of Gad8 (GenScript) was used to detect TORC2-dependent phosphorylation of Gad8 at S546. Total Gad8 was detected after incubation with a rabbit polyclonal antibody obtained after immunization with a peptide corresponding to N-terminal end of Gad8 (GenScript). Immunoreactive bands were revealed with anti-rabbit HRP-conjugated secondary antibody (Sigma) and the ECL system (GE-Healthcare).

NFs and KFs were divided into three groups Control (“C”, only treated with medium), Vehicle (“V”, medium 0.55% DMSO), and TA (550 μM TA dissolved in 0.55% DMSO with medium) for 48 h exposure separately. Proteins were extracted from each cell line and the concentrations were measured by BCA assay. Sixty micrograms of each sample were mixed with 10 μL of 5X protein sample loading buffer. The trans-blotted PVDF membrane was then blocked with blocking buffer (3% BSA; 1X TBS; and 0.05% Tween-20) for 1 h. Afterward, It was incubated with rabbit monoclonal anti-human collagen type I (dilution 1 : 2000, Thermo Fisher Scientific, USA), over two nights at 4°C. Anti-rabbit HRP-conjugated secondary antibody (dilution at 1 : 20000, Sigma, USA) was then added to the membrane on a shaker for an hour at room temperature. After rinsing with TBST, the membrane was activated with chemiluminescent HRP substrate (Cell Signaling Technology, USA) for 4 min in the dark at room temperature. It was visualized and quantified using a chemiluminescent detection system (Bio-Rad, USA). Protein band intensity in each lane was scored by volume intensity and was normalized to beta-actin (dilution at 1 : 4000, Sigma, USA) [27 (link)]. All experiments were performed independently. Four times with keloid fibroblast cultures culture as well as normal skin dermal culture.

Recombinant nucleoprotein, Fruend’s complete and incomplete adjuvants (Sigma, USA), RPMI 1640 media and Bovine serum albumin (Thermo Fisher Scientific, USA), IgG purification kit, anti-mice HRP, anti-rabbit HRP conjugated secondary antibody (Sigma, USA), HRP-conjugation kit (Abcam, USA), Maxisorp plate (Nunc, Denmark), Mouse IgG isotyping kit and 3,3,5,5-tetramethylbenzidine (TMB) (Invitrogen, USA).

Protein samples were separated by 10% Glycine SDS-PAGE, transferred to Amersham Protran Premium NC Nitrocellulose Membranes (GE Healthcare Life Sciences) and blocked for one hour under gentle stirring at room temperature with 50 mL of blocking solution (5% BSA in phosphate buffered saline [PBS] plus 0.2% Tween 20 [PBS-T]; Sigma). Fibronectin was detected by overnight incubation at 4 °C with a rabbit polyclonal primary antibody (Abcam) diluted 1:5,000 in PBS-T with 1% BSA and subsequent incubation for 2 h at room temperature with an anti-rabbit HRP-conjugated secondary antibody (Sigma) diluted 1:8,000 with PBS-T. Blots were incubated with mild stripping buffer (1.5% glycine pH 2.2, 0.1% SDS, 1% Tween 20) and further washed with PBS and PBS-T under gentle agitation at room temperature. Blots were re-blocked and re-probed.
His₆-tagged proteins were detected with the monoclonal His-HRP Conjugated Antibody (Santa Cruz Biotechnology) diluted 1:5,000 in PBS-T with 1% BSA incubated overnight at 4 °C and subsequently visualized with an enhanced chemiluminescence system (Super Signal West Pico Chemiluminescent; Pierce) according to the manufacturer’s instructions. Membranes were exposed to Hyperfilm ECL films (GE Healthcare Life Sciences).

The corpus callosum was homogenized in a RIPA buffer containing a mixture of protease inhibitors (Roche Applied Science, Penzberg, Germany). Twenty micrograms of protein was mixed with 5 × Laemmli buffer, then denatured for 5 min at 60 °C, separated by 10% SDS-PAGE, and finally transferred onto nitrocellulose membrane (Amersham Biosciences, Little Chalfont, UK). The blocking step was performed in 5% (w/v) non-fat dry milk in TBS with 0.1% Tween 20 (v/v) (TBS-T). The membrane was incubated in 1% (w/v) non-fat dry milk in TBS-T using the following antibodies: rabbit anti-CB₁ receptor primary antibody (1:500; Frontier Science, Hokkaido, Japan) and rabbit anti-β-actin (1:3000; Sigma-Aldrich, Hamburg, Germany). All primary antibodies were recognized by the anti-rabbit HRP-conjugated secondary antibody (1:1500; Sigma-Aldrich, Hamburg, Germany) followed by ECL-detection (Bio-Rad, Hercules, USA). The grouping blots were cropped from different parts of the same gel of apparently irrelevant lanes and and exposed exactly the same way. Chemiluminescence was identified by Amersham HyperfilmTM ECL (GE Healthcare, Little Chalfont, UK) and then quantified with Image J software (National Institutes of Health, Bethesda, USA). N = 4mice/group.

Twenty micrograms of protein was resolved by 8% SDS–PAGE and transferred onto a nitrocellulose membrane. The membrane was blocked in PBS 10% w/v nonfat dry milk at RT with gentle shaking for 2 h. The membrane was incubated with anti-iNOS (rabbit polyclonal IgG, 1:10,000 Sigma-Aldrich), anti-COX-2 (rabbit polyclonal IgG, 1:2000 Cell Signaling), anti-NF-κB p65 (clone 1G10.2, 1:500) mouse monoclonal antibody (Sigma-Aldrich), and anti-GAPDH HRP-conjugated (1:50,000) primary antibodies, at 4 °C. The blots were washed three times and incubated with anti-rabbit HRP-conjugated secondary antibody (Sigma-Aldrich) 1:80,000 or anti-mouse HRP-conjugated secondary antibody (Sigma-Aldrich) 1:50,000 for 1 h, RT. After washing three times, immunoreactive bands were detected using ECL (LuminataCrescendo, Merck Millipore, Burlington, MA, USA) and images acquired by LAS4000 (GE Healthcare, Chicago, IL, USA). The optical densities of immunoreactive bands were analyzed by ImageQuantTL software (GE Healthcare, Chicago, IL, USA, V 7.0) using GAPDH as a loading normalizing factor.