Anti rabbit igg antibody

We isolated extracellular and whole cell proteins from 1-day-old biofilms in accordance with a previous protocol^{33 (link)}. The culture supernatants were collected via centrifugation at 5000 × g for 5 min and incubated with 20% trichloroacetic acid. After washing with ice-cold acetone, the precipitates were dissolved in SDS sample buffer (50 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 2.5% mercaptoethanol, and 0.1% bromophenol blue). The cells were incubated with 1 μg ml⁻¹ lysozyme in PBS for 5 min and then dissolved in SDS sample buffer. After boiling for 10 min, the protein samples (OD₆₀₀ = 0.002) were separated by SDS-PAGE and electroblotted onto PVDF membranes. The membranes were blocked with 2.5% skim milk in Tris-buffered saline containing 0.05% Tween 20. BsaA and FLAG proteins were probed with anti-BsaA and anti-DYKDDDK antibodies (Wako) diluted 1:5000. These antibodies were labeled with anti-rabbit IgG antibodies (GE Healthcare) diluted 1:20000. The bound antibodies were labeled with Immunostar LD (Wako, Osaka, Japan) and detected using a FUSION-SL7-400 Chemiluminescence Imaging System (Vilber-Lourmat, Marne-la-Vallée, France). All the blots presented as part of the same series were derived from the same experiment and were processed in parallel. Original blots are shown in Supplementary Information.

Anti-GFP (Sigma-Aldrich, St Louis, MO, USA; F3165, 1:5000 dilution), anti-GST (Sigma-Aldrich; 1:5000 dilution), anti-LH (NEB, 1:3000 dilution) and anti-HYL1 (Agrisera, 1:1000 dilution) antibodies were used for Western blotting. The secondary antibodies used were goat-developed anti-rabbit IgG antibodies (GE Healthcare; NA931V, 1:20 000 dilution).

HEK293T cells were lysed in 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 50 mM NaF, 0.5% Nonidet P-40, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin A, and the supernatants were collected after centrifugation. Immunoprecipitation and immunoblotting were performed using standard procedures. anti-Flag antibody (1 μg; clone M2, Sigma-Aldrich, St. Louis, MO, USA) was used for immunoprecipitation. Primary antibodies for immunoblotting were: anti-ubiquitin (1:1,000; clone P4D1, Cell Signaling Technology, Danvers, MA, USA), anti-Flag (1 μg/ml; clone M2), and anti-α-tubulin (1:2,500; Abcam, Cambridge, MA, USA) antibodies. Secondary antibodies were peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG antibodies (GE Healthcare). Blots were detected using ECL Western Blotting Detection Reagents (GE Healthcare) and the ImageQuant LAS 4000 mini chemiluminescence detection system (GE Healthcare).

Proteins in each sample were denatured by boiling for 5 min in Laemmli’s sample buffer and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were electrically transferred onto Immobilon-PVDF membranes (Millipore, Bedford, MA), and membranes were blocked for 2 h with 5% non-fat dry milk-containing TBST (0.1% Tween-20, 100 mM NaCl, 10 mM Tris-HCl, ph 7.6). Primary antibodies against PrP (6D11, COVANCE, Dedham, MA; SAF32, Cayman Chemical Company, Ann Arbor, MI; 3F4, BioLegend, San Diego, CA), pro-caspase 3 (Cell Signaling, Beverly, MA), the cleaved caspase 3 (Cell Signaling), NP (GeneTex), NS1 (GeneTex), HA (GeneTex), M2 (GeneTex), podoplanin (MBL), SP-C (Santa Cruz Biotechnology), CC10 (Santa Cruz Biotechnology), XO (Santa Cruz Biotechnology), SOD1 (Abcam, Cambridge, UK), SOD2 (Abcam) and β-actin (Sigma-Aldrich) were incubated with the membrane overnight at 4°C. Signals were visualized using HRP-conjugated anti-mouse IgG antibodies (GE Healthcare), anti-rabbit IgG antibodies (GE Healthcare), anti-rat IgG antibodies (GE Healthcare), or anti-goat IgG antibodies (R&D systems, Minneapolis, MN), and detected using a chemiluminescence image analyzer LAS-4000 mini (Fujifilm Co., Tokyo, Japan). Signal intensities were measured using ImageJ 64.

The following antibodies are used: Lyn (clone 9, Wako; H-6, sc-7274, Santa Cruz Biotechnology), actin (C4, Millipore; 13E5, Cell Signaling Technology), phosphotyrosine (pTyr) (4G10, Upstate Biotechnology Inc.), Src (#327, Calbiochem), anti-Src[pY⁴¹⁶] (phospho-Src family, Cell Signaling Technology), cyclin B1 (H-433, Santa Cruz Biotechnology; V152, Cell Signaling Technology), phospho-histone H3 serine at position 10 (H3pS10) (clone 6G3, #9706 S, Cell Signaling Technology), and α-tubulin (MCA78G, Serotec). Horseradish peroxidase (HRP)-F(ab′)₂ fragments of anti-mouse IgG antibody, anti-rabbit IgG antibody, and of anti-rat IgG antibody, and Alexa Fluor 488-donkey-anti-mouse IgG antibody were purchased from GE Healthcare, Cell LAB, and Invitrogen.

To collect protein from in vivo retinas, mice were deeply anesthetized and euthanized by inhalation overdose of CO₂ 2 weeks after virus injection, and after retinal dissection, proteins were extracted in radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific, Waltham, MA, USA). For Western blots, the samples were boiled at 100 °C for 5 min, and equal amount of proteins from cell lysates were loaded on the SDS PAGE. Then proteins were transferred to PVDF membrane and incubated with primary antibody at 4 °C overnight. Primary antibodies used for these experiments included anti-Elk-1 (1:1000, ab131465; Abcam, Cambridge, UK, sc-365876; Santa Cruz Biotechnology), anti-Flag (1:1000, 14793; Cell Signaling Technology), anti-GAPDH antibody (1:2000; Cell Signaling Technology). Secondary antibodies were horseradish peroxidase (HRP)–conjugated anti-mouse IgG (1:2000; NA-931; GE Healthcare Life Science, Little Chalfont, United Kingdom) and anti-rabbit IgG antibody (1:3000; NA-934; GE Healthcare Life Science). Immunopositive bands were visualized with enhanced chemiluminescence (ECL; Thermo Fisher Scientific) and imaged on an LAS-3000 (Fujifilm, Tokyo, Japan). Densitometry was performed with ImageJ (http://imagej.nih.gov/ij/; NIH, Bethesda, MD, USA).