Pdquest analysis software

Endometrial and endometriotic tissues were suspended in RIPA⁺⁺⁺ buffer for Sodium Dodecil Sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Pooled samples were obtained by mixing equal protein amounts (μg of total protein) of 5 individual samples that were extracted in the same lysis buffer. The protein concentration in pooled samples was re-estimated using the appropriate quantification protocol. About 25 ng per sample were run on 12% SDS-PAGE for electrophoresis separation and then transferred to nitrocellulose membranes in a mini Trans-Blot apparatus (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were subsequently blocked with 3% w/v non-fat dry milk in 10 mMTris–HCl (pH: 7.5), 0.15 M NaCl, for 1 h at room temperature and then probed overnight at 4 °C with primary polyclonal antibody as reported in Supplementary Table S2, diluted in 1% non-fat dry milk/TTBS (TBS containing 0.2% Tween 20). After washing, membranes were incubated for 1 h with the appropriate horseradish peroxidase-conjugated secondary antibody. Immunoreactivity was detected by using chemiluminescence reagents (HRP Immuno-Star HRP substrate kit; Bio-Rad Laboratories, Hercules, CA, USA). The chemiluminescence signals were captured using a Bio-Rad Chemi-Doc system and quantified using a PDQuest analysis software (Bio-Rad Laboratories, Hercules, CA, USA).

Images of the 2DE Western blots were saved as TIFF files for analysis using Quantity One 1-D and spot identification with PDQuest analysis software (Bio-Rad). Protein density was quantified by dividing each membrane into a 7 x 23 grid of 161 equal sized rectangles that were placed on the identical positions on each individual image of the 2DE Western blots based on pI and MW. The average density of each rectangle was used to determine whether Sp185/333 proteins with varying pI identified in multiple sea urchins differed in prevalence within a given MW region on a blot. Duplicate samples from the same sea urchin were run and analyzed in parallel to ensure that the observed shifts were not artifacts of protein spreading in 2DE that resulted from sample processing (S1 Protocol; S3 Fig).