Goat anti rabbit igg

4T1 cells were seeded at 1x10⁶ cells per 35 mm dish. Next day, the cells were infected for 1 h at the indicated MOI of Ad virus. Following a 48 or 72 h incubation, whole-cell lysates were collected using 2 × SDS/PAGE protein loading buffer (62.5 mm Tris HCl pH 6.8, 25% glycerol, 2% SDS, 0.01% bromophenol blue, 5% β-mercaptoethanol). Samples were boiled for 5 min, separated by electrophoresis on a 15% SDS-polyacrylamide gel, and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Etobicoke, ON, Canada). The resulting membrane was probed with a mouse HA tag monoclonal antibody (1:10 000, Cell Signaling (Beverly, MA, USA) #2367), rabbit cleaved caspase-3 monoclonal antibody (1:1000, Cell Signaling #9664) or full-length caspase-3 antibody (1:1000, Cell Signaling #9662) and 1:5000 goat anti-rabbit IgG (Bio-Rad, Mississauga, ON, Canada, #170-6515) or goat anti-mouse IgG antibody conjugated to horseradish peroxidase (HRP) (1:10 000, Bio-Rad #170-6516). The membrane was also probed with antibody to α-tubulin to confirm equal loading (1:5000 rabbit α-tubulin antibody, AbCam (Toronto, ON, Canada) #ab15246, 1:5000 goat anti-rabbit IgG conjugated to HRP, Bio-Rad #170-6515). Blots were developed using the Pierce Enhanced Chemilumescent (ECL) Western Blotting Substrate (Thermo Scientific, Waltham, MA, USA).

Salivary glands (20 glands per well) were separated by SDS-PAGE on 12% polyacrylamide gels under denaturing conditions using the Mini-ATTO apparatus (Biogen). Proteins were either stained by Coomassie brilliant Blue (Bio-Rad) or transferred to PVDF membranes that were cut into strips (area corresponding to one well of the 12 well-comb was cut into 4 strips) and blocked overnight at 4°C with 5% milk in Tris-Tw buffer (20 mM Tris, 150 mM NaCl, 0.1% Tween^® 20; pH 7.4) at 4°C. After washing in Tris-Tw, membranes were incubated for 1 h with diluted sera (mice: 1:100; rabbit: 1:50). The strips were further washed and incubated with peroxidase conjugated goat anti-mouse IgG (1:500, AbD Serotec) or goat anti-rabbit IgG (1:2.500, AbD Serotec). Immunogenic protein bands were visualized by substrate solution with diaminobenzidine and reaction was stopped with distilled water.

Specific anti-P. perniciosus saliva antibody response was measured by indirect ELISA, according to Rohoušová et al. with minor modifications [21 (link)]. Sera were diluted (1:50) in 2% skimmed milk and incubated at 37°C during 3 h. For mouse IgG subtype studies (IgG and IgG2a) sera were diluted 1:200. Peroxidase-conjugated antibodies were incubated for 1 h at 37°C (goat anti-mouse IgG 1:500, goat anti-mouse IgG1 1:10.000, goat anti-mouse IgG2a 1:1.000, goat anti-rabbit IgG 1:2.500; AbD Serotec and goat anti-rabbit IgM 1:20.000; Bethyl Laboratories). Plates were developed with orthophenylendiamine in McIlwein phosphate-citrate buffer (pH 5.5) in the presence of H₂O₂. The reaction was stopped with 10% H₂SO₄ and absorbance was measured at 492 nm using an ELISA reader (ELx800, BioTek, Winooski, VT). Each serum was tested in duplicate. The control wells were coated with SGH, but no serum was added. IgG antibody levels were reported as adjusted optical density (aOD), calculated for each serum as a mean OD value of the duplicated wells minus the OD value of the control wells. ELISA experiments were repeated at least twice.

We used 30–50 μg of microsomal protein for western blots to measure CYP levels. Proteins were separated by polyacrylamide gel electrophoresis (SDS-PAGE) in a 10% gel, and transferred to a 0.45-μm polyvinylidene fluoride (PVDF) membrane, then the blot was blocked with 5% skim milk and 0.3% Tween 20 dissolved in phosphate-buffered saline (PBS). The membrane was washed in PBS three times for 10 min and incubated for 1.5 h at 37°C with primary rabbit anti-mouse antibodies for recognizing CYP1A1/2 and CYP2B (clone 14H5 and 12F10, respectively), which were obtained from Dr Grishanova [37 ] and diluted 1:20 in TBS. A rabbit anti-mouse β-actin primary antibody (diluted 1:500; Sigma-Aldrich, St. Louis, MO) was used to ensure equal loading of samples. Goat anti-rabbit IgG (Bio-Rad) alkaline-phosphatase-conjugated secondary antibodies were used to detect the primary antibodies to CYP1A1/2, CYP2B, and β-actin. The protein bands were visualized colorimetrically with alkaline-phosphatase fastTM Tablets (Sigma-Aldrich) as substrates, according to the manufacturer’s directions. Colorimetric blots were scanned on a Bio-Rad VersaDoc 4000 Imaging System (Bio-Rad), and the bands were quantified in the ImageJ software (NIH, Bethesda, MD, USA). The experiments were performed using equipment of center of “Proteomic analysis” of the Institute of Molecular Biology and Biophysics (Novosibirsk, Russia).

Glibenclamide (G0639), Gly-Phe (G2752), and Gly-Sar (G3127) were purchased from Millipore Sigma. Glycyl-L-proline (Gly-Pro, 405935) was purchased from Frontier Scientific. Puromycin dihydrochloride (A11138), Pierce Western Blot reagents, and Applied Biosystems qPCR reagents were purchased from ThermoFisher Scientific.
The antibodies specific for hSLC15A1 and hSLC15A2 were generously supplied by David E. Smith, College of Pharmacy, University of Michigan. Other antibodies used were: anti-β-actin (Santa Cruz Biotechnology, sc-47778), anti-LAMP-1 (Abcam, ab25630), anti-MMP9 (Cell Signaling, D6O3H), anti-MMP13 (Cell Signaling, E4W3T), anti-MMP16 (Abcam, ab73877), anti-DPP4/CD26 (Cell Signaling, D6D8K), goat anti-rabbit IgG (Bio-Rad, 170-6515), goat anti-mouse IgG (Bio-Rad, 170-6516), goat anti-mouse IgG alexa fluor 488 (ThermoFisher, A-11029), goat anti-rabbit IgG alexa fluor 594 (ThermoFisher, A-11037), goat anti-rabbit IgG alexa fluor 488 (Life Technologies Corp, A11008), goat anti-mouse IgG alexa fluor 647 (Life Technologies Corp., 647), and ProLong Diamond Antifade Mountant with DAPI (Life Technologies Corp., P36962),