Nitrocellulose filter membrane

AGL1 carrying pHMGB or pRp27GFP-NLS was spread on an LB agar plate containing kanamycin (50 mg/L) and incubated for 2 days at 28°C. A single colony was cultured in 5 mL of LB broth containing 50 mg/L kanamycin at 28°C with a shaking speed of 200 rpm for 48 h. Cells of 2 mL culture were then collected by centrifugation (5,000 × g) for 10 min, washed using A. tumefaciens induction medium (AIM, lipid) for 2 min, and then diluted, respectively, into OD₆₀₀ about 0.6 in AIM supplemented in 200 mM acetosyringone (AS) and an AS-free AIM as a control. For transformation, 100 μL of A. tumefaciens cells were mixed with 100 μL 1 × 10⁶ of A. alternata conidia, the mixture was spread on a sterilized nitrocellulose filter membrane (Pore size 0.45, Φ50 mm, Whatman, Sangon, Shanghai, China) overlaid on the surface of AIM agar plates and incubated for another 48 h at 23°C in the dark. After the incubation, the nitrocellulose filter membrane was cut with a sterilized knife into strips and transferred into selective CM plates containing 200 μg/mL hygromycin B, 200 μg/mL cefotaxime sodium, 200 μg/mL tetracycline hydrochloride and incubated for 5–7 days to selected the transformants. Thirteen selected hygromycin B-resistant transformants were verified by growing in a new selective CM plate for another 3–4 days at 28°C, together with the wild-type as a control.

Protein aliquots were electrophoresed on SDS PAGE and then transferred to nitrocellulose filter membrane (Whatman, Dassel, Germany). The blots were blocked with 5% skim milk in TBST (10 mM Tris Base, 150 mM NaCl, 0.1% Tween-20, pH 8.0) for 1 hour at room temperature, and incubated overnight with primary antibodies. Rabbit anti-human IgG γ Fc region antibody (Dako, Copenhagen, Denmark), mouse anti-human Ig κ chain antibody (ZSGB-BIO, Beijing, China) and mouse anti-human Ig λ chain antibody (ZSGB-BIO, Beijing, China) at a dilution of 1:1,000 were used as primary antibodies. Goat anti-rabbit IgG-680 (1:10,000; LI-COR, Nebraska, USA) and goat anti-mouse IgG-680 (1:10,000; LI-COR, Nebraska, USA) were used as secondary antibodies. The blots were examined with a Odyssey imaging system (LI-COR, Nebraska, USA).

Western blot analysis was performed as described in Wang et al. (2011) (link). The total protein was extracted by using the Plant Protein Extraction Kit (CWBIO, China) from 20-days-old seedlings grown in NP conditions. The total protein (60 μg) was separated on a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose filter membrane (Amersham, USA). The membrane was blocked with 5% milk in phosphate-buffered saline. The primary antibody [HA Tag monoclonal antibody (1:5000, Thermo Fisher, United States)] and the secondary antibody [goat anti-mouse IgG-HRP (1:3000, Sangon, China)] were used for the Western blot. The blotted membrane was incubated with ECL luminous solution MaxiLumin-WB (Biokits tech Inc., China) and visualized using an Odyssey FC imaging system (LI-COR, United States).

Mouse splenocytes and cultured cells were lysed using RIPA buffer in the presence of protease inhibitor and phosphatase inhibitor. The BCA protein assay was used to quantify the protein concentrations. Then, the protein samples were separated by denaturing 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) at 70 V for 3 h. The proteins were transferred to a nitrocellulose filter membrane (Amersham, Little Chalfont, UK) at 250 mA for 2 h. The membranes were incubated with the block buffer (5% skim milk in a 0.1% tween-PBS solution) at room temperature for 1 h, and subsequently incubated with the primary antibodies overnight. The membrane was washed using PBST and PBS, followed by incubation of RDyeCW800-conjugated secondary antibodies for 1 h at room temperature and visualization using the LI-COR Odyssey imaging system (LI-COR, Lincoln, NE, USA).

The HeLa cells were first lysed using radioimmunoprecipitation assay (RIPA) buffer (i.e. 25 mM Tris–HCl (pH 7.6), 150 mM NaCl, 0.1% SDS, 1% sodium deoxycholate, 1% NP-40 and 1× protease inhibitors), followed by immunoblotting using standard protocol. In brief, the extract was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto nitrocellulose filter membranes (Whatman), then incubation with the respective antibodies. This was followed by incubation with either horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (Bio-Rad) or HRP-conjugated anti-mouse IgG antibody (Bio-Rad) for 2 h at room temperature. Enhanced chemiluminescence substrates (Luminata Crescendo, EMD Millipore) were then applied and the signals exposed to autoradiography film. The immunoblots were then quantified by densitometric analyses using ImageJ software. All antibodies used were purchased from commercial sources (for details, see Supplementary Data).

The fractions of IVIG and their fragments with or without PNGase F treatment were separated with 10% SDS-PAGE under reducing conditions. In brief, samples were denatured with heating at 100°C for 10 minutes in denaturing buffer (62.5 mM, pH 6.8 Tris-HCl solution containing 10% Glycerol, 2% SDS, and 10 mM DTT), and then loaded into 10% SDS-PAGE gel. The samples were first ran at 80 voltage for 30 minutes, followed by ran at 120 voltage for about 60min till the dye run to the bottom of the gel. After electrophoresis, the gels were stained with silver or transferred to nitrocellulose filter membranes (Whatman, Dassel, Germany) and examined with Western blot or Lectin blot were described below.