Amersham hybond p pvdf membrane

Arabidopsis rosette leaves were collected to extract total proteins or nuclear proteins. For total protein extraction, leaves were mixed with extraction buffer (100 mM Tris–HCl pH = 6.8, 2% SDS, 200 mM DTT, 20% glycerol, and 0.5 mM PMSF). Extraction of nuclear proteins was performed employing the Plant Nuclei Isolation/Extraction Kit (CelLytic™ PN, Sigma-Merck, Darmstadt, Germany) following the manufacturer’s recommendations. Equal amounts of protein extracts were separated on SDS-polyacrylamide gels. The separated proteins were transferred onto Amersham Hybond-P PVDF Membrane (GE Healthcare, Chicago, United States) for 1 h at 100 volts. Membrane was blocked with TBST buffer, 0.1% Tween 20, and 5% skim milk for overnight at 4°C. Blots were incubated with antibody against ANAC087 (Genscript; Piscataway, NJ, United States) (1:2500) for 4 h at 4°C, washed, and incubated with a secondary antibody against rabbit IgG (whole molecule; Sigma-Merck) conjugated to goat peroxidase (1:10,000) for 2 h at room temperature. The washed blots were transferred to ECL Prime Western Blotting Detection Reagent (Pierce, Rockford, IL, United States) and then exposed to X-ray film.

Proteins were extracted using Complete Lysis-M (Roche, Indianapolis, IN, USA). Extracts were subjected to SDS-PAGE (Super Sep Ace 7.5, 10 or 15%, Wako Pure Chemical Industries, Osaka, Japan) and transferred onto a membrane (Amersham Hybond-P PVDF Membrane, GE Healthcare, Buckinghamshire, UK). Primary and secondary antibodies are listed above in the Reagents subsection. Antibody-protein complexes were detected using Immun-Star™ AP substrate (Bio-Rad Laboratories), and protein bands were visualized using an ImageQuant™ LAS 4000 image analyzer (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).

Western blot analyses were done as described previously [24 (link)]. Cell extracts were subjected to lysis in ice-cold buffer. The protein concentrations were determined, and the proteins were separated on a 10% sodium dodecyl sulfate polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (Amersham Hybond-P PVDF Membrane; GE Healthcare). Afterward, the membranes were blocked with 5% nonfat milk in 1×tris-buffered saline solution with 0.1% TBST for 1 hour at room temperature and washed with 1×TBST three times. They were incubated with the appropriate primary antibody overnight at 4°C and then with the appropriate secondary antibody for 1 hour at room temperature on a rocking platform. Expression of various proteins, including stage-specific embryonic antigen-1 (SSEA-1) and H-ras, was measured by using mixed ECL Plus reagents (RPN2132OL/AK, GE Life Sciences Co.) and developed by using an X-OMAT 2000 film processor (Kodak). HSP90 was used as a protein-loading control. The antibodies used are described in Table S2.

Expression of the target protein was compared between uninduced and induced RNAi cell cultures using cell lysates corresponding to 5 x 10⁶ cells per lane. Lysates were prepared in NuPAGE^® LDS sample buffer (Invitrogen), separated on Bolt 4–12% Bis-Tris polyacrylamide gels (Invitrogen) and transferred to an Amersham Hybond P PVDF membrane (GE Healthcare) for probing with the respective antibodies. Rat anti-alpha MPP, and rabbit anti-MIP were used at dilutions 1:1,000, 1:1,000 and 1:5,000, respectively. Polyclonal anti-enolase [48 (link)] and anti-trCOIV antibodies [49 (link)] were used at dilutions 1:50,000 and 1:10,000, respectively. Monoclonal anti-mtHsp70 was used at a dilution of 1:5000. Monoclonal anti-V5 (Invitrogen), polyclonal anti-GFP (Life Technologies) and monoclonal anti-tubulin antibodies (Sigma-Aldrich) were used at dilutions 1:2,500, 1:2,000 and 1:5000, respectively. Secondary antibodies were conjugated to horseradish peroxidase (Sigma-Aldrich), and the signal was visualized using Clarity Western ECL Blotting Substrate (Bio-Rad).

Ten adult fly heads were homogenized in 50 µl RIPA buffer on ice. They were centrifuged at 4°C for 20 min at 13,000 rpm. The supernatant was mixed with 5× reducing Lämmli buffer and incubated for 5 min at 65°C. Fifteen µl of the samples were separated to an 8% SDS-gel and subsequently blotted onto a PVDF membrane (Amersham Hybond-P PVDF Membrane, GE Healthcare). Anti-Para antibodies were generated against the following N-terminal sequence (CAEHEKQKELERKRAEGE), affinity purified, and were used in a 1/1000 dilution. Experiments were repeated three times.

The tissue was ground in liquid nitrogen, and total protein was extracted using 2×SDS loading buffer. The samples were resolved on a 12% SDS-PAGE gel and transferred onto an Amersham Hybond-P PVDF membrane (GE Healthcare) using Tris-Gly transfer buffer. Membranes were blocked with 5% (w/v) milk in 0.05% (v/v) TBS-plus Tween 20 (TBST) for 40 min and incubated overnight at 4°C with 1:5,000 dilutions of primary antibodies of mouse anti-Flag conjugated to horseradish peroxidase (Abmart), washed three times with TBST, and incubated overnight at 4°C with 1:1,000 dilutions of secondary antibodies (Beyotime). The membranes were then washed three times with TBST. Detection was performed using ECL Plus western blotting Detection Reagents (GE Healthcare) and ChemiDoc Touch Imager (Bio-Rad). Anti-GFP antibody (Abmart) was used to determine the expression level of the YFP protein. Ponceau was used as a loading control, and MAPK assays were performed as previously described (Zhang et al. 2021 (link)). Briefly, 1 mm of flg22 peptide was infiltrated into the leaves of 4-week-old plants. Total protein samples were collected at 0, 5, 10, 20, and 30 min and used for immunoblotting with an anti-p44/42 mAPK antibody (Cell Signaling Technology) to detect activated forms of MPKs. Image data were analyzed using Image Lab Software (Bio-Rad) and assembled using Adobe Photoshop CS6.