Protein extraction and immunobloting

TMEM95 is a sperm membrane protein essential for mammalian fertilization 

Protein extraction and Immunobloting

WT and KO sperm were collected from cauda epididydimis in PBS supplemented with 0.1 % PVP and centrifuged at 3,000 g for 7 min. Pellets and tissues samples (testis and accessory glands) were snap frozen in liquid nitrogen and kept at -80 ºC until analysis. 
 

a) For the analysis, frozen pellets were re-suspended in reducing SDS Sample Buffer (4X) (Millipore) and boiled for 10 minutes, centrifuged at 20.000 g at 4 ºC for 10 min and the supernatants were used. For membrane protein extractions, frozen pellets were re-suspended in 1 % Octyl β—D-Glucopyranoside (Sigma) solution in PBS, incubated on ice for 30 min and centrifuged at 20.000 g at 4 ºC for 10 min. The supernatants were used for the experiments (Nishimura et al. 2011). 
 

b) Cell suspensions from testis and accessory glands were obtained by thoroughly mincing tissues with razor blades. The cell pellet was re-suspended in 500 µl of 50 mM Tris-HCl ph 7.5, 1 mM EDTA, 1 % Igepal, 0.1 mM PMSF, 10 mM iodoacetamide, 10 mM N-ethylmaleimide, phosphatase inhibitor and protease inhibitor, homogenized and incubated for 30 min on strong agitation at 4 ºC and then centrifuged at 20,000 g for 20 min at 4 ºC (Abrisqueta et al. 2018). The supernatants were used for the experiments
 

The supernatants were separated in 16% SDS-PAGE and the proteins were transferred to PVDF membranes. The membranes were blocked with 5% BSA in TBST 1X for 1 h at RT, incubated with anti-TMEM95 (MBS7004333, MyBioSource, USA,) (1:1000 v/v in TBST 1X, 1% BSA), anti-IZUMO1 antibody (ab211623, abcam, Cambridge, UK) (1:1000 v/v in TBST 1X, 1% BSA) or Anti-β-Tubulin antibody (T8328, Sigma-Aldrich, Madrid, Spain) (1:5000 v/v in TBST 1X, 1% BSA) overnight at 4ºC. The next day the membranes were washed three times for 10 min with TBST 1X, incubated with corresponding peroxidase-conjugated secondary antibody and washed three times for 10 min with TBST 1X prior to visualization by chemiluminescence (Pierce ECL-Plus, Thermo Fisher Scientific). 

The bands corresponding to TMEM95 protein (⁓ 20 kDa) was cut out and processed for proteomic analysis. Data processing was performed with Data Analysis program for LC/MSD Trap Version 3.3 (Bruker Daltonik, GmbH, Germany) and Spectrum Mill MS Proteomics Workbench (Rev A.03.02.060B, Agilent Technologies, Santa Clara, CA, USA) by the Molecular Biology Section, Service of Support to the Experimental Sciences (SACE), University of Murcia. 

The bands were quantified by densitometric scanning and analysed with the ImageQuant TL software v2005 (GE Healthcare Buckinghamshire, UK). Izumo1/Tubulin ratio was calculated in arbitrary units with data from sperm from 4 WT and 4 KO animals. The obtained data was subjected to Student’s t-test and the level of significance was set at p < 0.05. The software used was IBM SPSS Statistics (v22.0).

Abrisqueta, Marta, Concepcion Olivares, Cecilia Herraiz, Maria Castejon-Grinan, Julia Sires-Campos, Jose C Garcia-Borron, and Celia Jimenez-Cervantes. 2018. “Human Melanocortin 1 Receptor-Mediated Ubiquitination of Nonvisual Arrestins. Role of Mahogunin Ring Finger 1 E3 Ligase.” Biochimica et Biophysica Acta. Molecular Cell Research 1865 (1): 76–94. https://doi.org/10.1016/j.bbamcr.2017.09.013.

Nishimura, Hitoshi, Surabhi Gupta, Diana G Myles, and Paul Primakoff. 2011. “Characterization of Mouse Sperm TMEM190, a Small Transmembrane Protein with the Trefoil Domain: Evidence for Co-Localization with IZUMO1 and Complex Formation with Other Sperm Proteins.” Reproduction (Cambridge, England) 141 (4): 437–51. https://doi.org/10.1530/REP-10-0391.

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