Journal:
Article Title: Developmental control of sumoylation pathway proteins in mouse male germ cells a
doi: 10.1016/j.ydbio.2008.06.020
Figure Lengend Snippet: A) Expression of SAE2 (i), UBE2I (ii), PIAS2 (iii), PIAS4 (iv), SENP1 (v) and SENP2 (vi) in leptotene/ zygotene (L/Z), prepubertal pachytene (PP) and adult pachytene (P) spermatocytes, as well as in round spermatids (RS). i) Expression of SAE2 increases from leptonema/ zygonema to pachynema and decreases in round spermatids. ii) Two bands are detected using an antibody against UBE2I, a smaller band of ~ 18kDa, and a larger band of ~ 38kDa. Expression peaks in pachytene spermatocytes. iii) PIAS2 levels are highest in pachytene spermatocytes and round spermatids. iv) An antibody directed against PIAS4 shows increasing levels of the protein with male germ cell development. v) SENP1 levels increase from leptonema/ zygonema until pachynema, and remain elevated in round spermatids. vi) SENP2 is prominent in leptotene/ zygotene spermatocytes. Equal amounts of total protein (10 µg) were loaded in each lane. Apparent molecular weights are given in brackets below each protein name (in kDa), while appropriate molecular weight markers are indicated to the right of each panel (in kDa). Western blots were conducted on each of the three series of germ cells; representative results for one series are presented here. B) i) Gel electrophoresed under identical conditions but stained with GelCode Blue Stain demonstrating equal protein loading in each lane. ii) Membrane stained with India ink following transfer illustrating equal protein loading as well as transfer consistency. Equal amounts of total protein (10 µg) were loaded in each lane. Lane identification is the same as in A).
Article Snippet: Membranes were blocked in 5% non-fat dried milk diluted in PBS supplement with 0.1% Tween 20 (PBST) and were incubated with one of the following primary antibody diluted in blocking buffer: rabbit polyclonal anti-SAE2 (IMG-5111A, 1:5000; Imgenex, San Diego, CA); mouse monoclonal anti-UBE2I (610749, 1:10000; BD Transduction Laboratories, San Jose, CA); goat polyclonal anti-PIAS2 (sc-30879, 1:1000; Santa Cruz Biotechnology Inc.); rabbit polyclonal anti-PIAS4 (IMG-290, 1: 5000; Imgenex); rabbit polyclonal anti-SENP1 (IMG-521, 1:2500; Imgenex); and rabbit polyclonal anti-SENP2 (AP1232c, 1:2500; Abgent, San Diego, CA).
Techniques: Expressing, Molecular Weight, Western Blot, Staining
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