Grace s insect media

Whole ovaries, S9 follicles (∼30/lane), and S10B follicles (∼15/lane) were dissected in room-temperature Grace's insect media (Lonza, Walkersville, MD). Standard Western blotting techniques were used. The following primary antibodies were used at the indicated concentrations: mouse anti−Enabled (5G2; Goodman, C.; obtained from the DSHB), 1:200; rabbit anti−Pxt (affinity-purified polyclonal antibody generated against a synthetic peptide: CQIRQEHGRIDEVVN; GenScript, Piscataway, NJ), 1:5000; and mouse anti−α−tubulin T9026 (Sigma-Aldrich, St. Louis, MO), 1:500. The following secondary antibodies were used: Peroxidase-AffiniPure goat anti–mouse immunoglobulin G (H+L), 1:5000; and Peroxidase-AffiniPure goat anti-rabbit immunoglobulin G (H+L), 1:5000 (Jackson ImmunoResearch Laboratories, West Grove, PA). Blots were developed with SuperSignal West Pico or Femto Chemiluminescent Substrate (Thermo Scientific, Waltham, MA) and imaged using the ChemiDoc-It Imaging System and VisionWorksLS software (UVP, Upland, CA). Bands were quantified using densitometry analysis in ImageJ (Abramoff et al., 2004 ). Ena levels were assessed using a minimum of three independent biological samples. Statistical significance was determined using a two-sample t test with unequal variance in Excel (Microsoft, Redmond, WA).