Goat α mouse hrp

Primary antibodies employed were: anti-CD63 (Tea 3/10), and anti-CD9 (VJ1/20) mAbs (Immunostep)^{36 (link),37 (link)} conditioned media from mouse hybridoma; polyclonal anti-EEA-1 (Santa Cruz) (1:50 for immunofluorescence) and polyclonal anti-GFP (Living colours, Clontech) (1:1000 for immunoblotting, 1:200 for immunofluorescence).
Secondary Abs employed were Goat-α-Mouse Alexa647 (Life technologies), Donkey-α-Goat Alexa647, Donkey-α-Rabbit Alexa488 and Phalloidin-Alexa647 (Invitrogen) (1:200 for immunofluorescence); Goat-α-Mouse HRP and Goat-α-Rabbit HRP (Thermo scientific) (1:5000 for immunoblotting).

Western blotting was performed with polyclonal peptide antibodies against either Ryp1, Ryp2, or Ryp3; antibodies were described previously by Beyhan and colleagues [2 (link)]. Following quantification of protein, 10 to 30 μg was resuspended in a total of 20 μL of urea lysis buffer and 6 μL Novex NuPAGE LDS Sample Buffer (Invitrogen, Carlsbad, CA). The samples were boiled and electrophoresed on a 10-well Novex NuPAGE 4% to 12% BT SDS-PAGE gel (Invitrogen, Carlsbad, CA) in MOPS running buffer at 150 V. The protein was then transferred to a nitrocellulose membrane at approximately 40 V for 2 hours. The membrane was incubated with blocking solution (1 g milk powder in 100 mL wash buffer [0.1% Tween-20 in PBS]) for an hour and then incubated in the primary antibody in wash buffer overnight at 4 °C. Primary antibody dilutions were: α-Ryp1 (1:10,000), α-Ryp2 (1:2,500), α-Ryp3 (1:5,000), α-GAPDH (1:1,000). The blot was washed and secondary antibody (for Ryp proteins: Goat α-rabbit HRP [GenScript, Piscataway, NJ] 1:1,000, for GAPDH: Goat α-mouse HRP [ThermoFisher, Weltham, MA] 1:1,000) was added to the blot for 1 hour at RT followed by another wash. Protein bands were detected using chemiluminescence according to the manufacturer’s instructions (SuperSignal West Pico kit; ThermoFisher, Weltham, MA).

Mouse testis samples were lysed using RIPA buffer (Sigma Aldrich, St. Louis, MO, USA) containing a protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO, USA), and 10 µg/mL total protein was separated on 10% polyacrylamide gel via SDS-PAGE, followed by transfer on methanol-activated PVDF membrane (Merck Millipore, Burlington, MA, USA). The membranes were blocked in 5% non-fat powdered milk for 1 h at room temperature, after which they were incubated with primary antibodies to Akt (pS473) + total Akt Kit (ab126433, abcam, Cambridge, UK), ERK1 (phosphor) + total ERK (ab126445, abcam), p38 MAPK alpha + total p38 MAPK alpha (ab126453, abcam), cyclinD1 (ab16663, abcam) and β-actin (A5441, Sigma Aldrich, St. Louis, MO, USA), overnight, at 4 °C. The next day, after 3 washes with 0.1% PBST, the membranes were incubated with the corresponding secondary antibodies: goat α-mouse HRP or goat α-rabbit HRP (Thermo Scientific) for 1 h at room temperature. The membranes were developed with Immobilon Forte HRP Substrate (Merck Millipore, Burlington, MA, USA) using a Luminescent Image analyzer LAS-3000 (FUJIFILM, Tokyo, Japan). The relative protein expression was quantified via densitometric analysis with the Image J software (Version 1.53r).