Carbohydrate and other epitopes of contact site A glycoprotein of Dictyostelium discoideum as characterized by monoclonal antibodies
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Absence of catalytic domain in a putative protein kinase C (PkcA) suppresses tip dominance in Dictyostelium discoideum
2015, Developmental BiologyCitation Excerpt :Subsequently, the membrane was blocked with 5% BSA and 0.05% Tween-20 in Tris-buffered saline (TBS) for 1 h at RT. Later, the membrane was incubated overnight at 4 °C with primary antibodies anti-CadA (1:10,000) (Knecht et al., 1987) or anti-CsaA (1:10) (Bertholdt et al., 1985) or anti-actin (1:10) (Simpson et al., 1984). To remove unbound primary antibodies several washes were carried out with TBS–Tween-20, and the membrane was incubated with 1:5000 dilution of HRP conjugated secondary antibody (Bangalore Genei, India) for 1 h at RT.
The thyroxine inactivating gene, type III deiodinase, suppresses multiple signaling centers in Dictyostelium discoideum
2014, Developmental BiologyCitation Excerpt :Cell lysates were analyzed by electrophoresis in 10–12% polyacrylamide gels and were blotted onto a nitrocellulose membrane (Bio-Rad). The membranes were independently incubated with anti-CsaA (Bertholdt et al., 1985; Ochiai et al., 1982) (1:10; a kind gift from Ludwig Eichinger, University of Cologne, Germany), anti-CadA (Knecht et al., 1987) (1:10,000; a kind gift from Chi-Hung Siu, University of Toronto, Canada), anti-GFP (Mitra et al., 2003) (1:5000; Santa Cruz Biotechnology) and anti-actin (Simpson et al., 1984) (1:10; a kind gift from Angelika Noegel, University of Cologne, Germany) primary antibodies overnight at 4 °C. Membranes were washed thrice with TBST buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl and 0.1% Tween 20) for 30 min and thereafter, the membranes were incubated with secondary antibodies (1:4000) conjugated with horseradish peroxidase (HRP) for 1 h at RT.
The G alpha subunit Gα8 inhibits proliferation, promotes adhesion and regulates cell differentiation
2013, Developmental BiologyCitation Excerpt :Rabbit anti-tgrC1 antiserum (Geltosky et al., 1979) was kindly provided by Dr. Charles Singleton at Vanderbilt University. Monoclonal mouse anti-CsA antibody (33-294-17) (Bertholdt et al., 1985) was obtained from the Developmental Studies Hybridoma Bank at the University of Iowa. Monoclonal mouse anti-Actin antibody (MAB1501R) was purchased from Millipore.
ForC lacks canonical formin activity but bundles actin filaments and is required for multicellular development of Dictyostelium cells
2013, European Journal of Cell BiologyCitation Excerpt :Proteins were resolved by SDS-PAGE in 7.5%, 10% or 15% gels, and immunoblotting was performed by standard procedures using ForC polyclonal antibodies or DGAP1-specific mAb 216-394-1 (Faix and Dittrich, 1996). D19 (also referred to as PsA) was detected with Mud-1 monoclonal antibody (Gregg et al., 1982) and csA was probed with mAb 33-294-17 (Bertholdt et al., 1985). Primary antibodies were visualized with phosphatase-coupled anti mouse or anti-rabbit IgG (Dianova).
The Ras related GTPase Miro is not required for mitochondrial transport in Dictyostelium discoideum
2011, European Journal of Cell BiologyCitation Excerpt :Disruption of the gemA gene was verified using PCR, Southern blot and Western blot as described in Figure S1. Actin was detected using mAb Act 1-7 (Simpson et al., 1984), mitochondria using a mAb against porin (Troll et al., 1992), tubulin using mAb YL1/2 (Kilmartin et al., 1982) and mAb WA3 (gift from Dr. Ursula Euteneuer), contact site A using mAb 33-294-17 (Bertholdt et al., 1985), GFP using mAb K3-184-2 (Noegel et al., 1999) and DdKif5 using a specific antiserum (Iwai et al., 2004). The appropriate Cy3 or Alexa Fluor-labeled anti-mouse immunoglobulins (Molecular Probes) were used as secondary antibodies.
A Coronin7 homolog with functions in actin-driven processes
2010, Journal of Biological Chemistry