Quantity one software

For normalization in Western analysis, all fractions were compared on the basis of equal amounts of original starting material (e.g., 3-mg roots). This method of comparison was used because our aim was to analyze the yield of total membranes from a fixed amount of material. Also, to follow the sedimentation of membranes during successive centrifugation steps (e.g., preclearance and final), this was the only valid way of comparing the yield at each step from the same sample.
Membrane pellets were resuspended (0.5–1 μl/mg material) in storage buffer (20 mM Tris–HCl [pH 7.5], 5 mM EDTA, 5 mM EGTA, 20% glycerol, and protease inhibitors without casein) or solubilized directly into sample buffer (SB: 2% sodium dodecyl sulfate [SDS], 125 mM Tris–HCl [pH 6.8], 40 mM DTE, and either 20% glycerol or 8 M urea). If in storage buffer, 2- to 7-μl membranes (containing 2–10 μg protein) were added to 2× SB. Samples were left at room temperature or heated (50 °C, 10 min). The 600g pellets were extracted by heating in 2× SB (90 °C, 3 min). Samples were cleared (10,000g, 2 min) and separated by SDS–polyacrylamide gel electrophoresis (PAGE) [23] (link) with 3 M urea in the stacking gel.
Gels were blotted and Western analysis was performed using standard procedures. Detection was performed using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence substrate (SuperSignal Pico or Femto, Pierce). Blots were exposed to X-ray film and/or recorded on a ChemiDoc XRS Imager (Bio-Rad). Quantitation was performed only for the latter using Quantity One software (Bio-Rad). Comparisons and quantitation of data were performed only between samples that were run on the same blot. The presence of a white space between the lanes in a panel (e.g., Fig. 3) indicates that the samples were on the same blot but were not in contiguous lanes. Figures presented here have been cropped from full-length blots, examples of which are presented in Fig. S1 in the supplementary material. All experiments were repeated at least twice, and the UC and preclearance validations were performed four to six times.

After harvesting, the cells were lysed in 50 mM Tris-HCl (pH 8.0) containing 150 mM NaCl, 1% nonidet P-40, protease inhibitor cocktail, 1 mM phenylmethane sulfonyl fluoride, 25 mM sodium fluoride, and 1 mM sodium orthovanadate. Protein concentrations were determined using BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Subsequently, cellular extracts (45~60 μg) were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane. Non-specific binding was blocked with 5% skim milk in Tris-buffered saline with Tween 20 for 30 min at room temperature. The membranes were then incubated with the primary antibody (1 : 1,000) overnight at 4°C and continually with the secondary antibody (1 : 5,000) at room temperature for 30 min. After incubation, the membranes were washed three times for 10 min each. Proteins were visualized using ECL reagent, and the band intensity was measured using a Chemidoc XRS densitometer system and quantified by Quantity One software (Bio-Rad).