Hpa011924

Mouse tissues and cell lysates were extracted using RIPA lysis buffer (20–188, Millipore) according to the manufacturer’s protocol. Cell culture supernatant was collected and concentrated with Amicon Ultra-15 tubes (10 KDa, Millipore). The following antibodies were used: anti-RRBP1 (PA5-21,392, Invitrogen), anti-RRBP1 (Ab95983, Abcam), anti-RRBP1 (HPA011924, Sigma), anti-Hsp70 (DF2698, Affinity), anti-β-actin (GTX109697, Genetex), anti-renin (H0005972-M01, Abnova), anti-ACE (MA5-32,741, Invitrogen), anti-β-tubulin (tcaba2, Taiclone), anti-SGK1 (ab32374, Abcam), anti-ADCY5/6 (PA5-75,274, Abnova), and anti-calnexin (ab22595, Abcam). The blots were detected with Trident pico Western HRP Substrate (GTX17435, Genetex), and images were analyzed using a UVP BioSpectrum Imaging System.

SuperSignal West Pico Chemiluminescence Susbtrate (# 34078) was purchased from Pierce^TM, Thermo Fisher Scientific, Darmstadt, Germany. ECL^TM Plex goat anti-rabbit IgG-Cy5 (PA45011, used dilution 1:1000), and ECL^TM Plex goat anti-mouse IgG-Cy3 conjugate (PA43009, used dilution 1:2500) were purchased from GE Healthcare, Freiburg, Germany. Horseradish peroxidase coupled anti-rabbit IgG from goat (A 8275, used dilution 1:1000) was from Sigma-Aldrich, Taufkirchen, Germany. We also purchased from Sigma-Aldrich murine monoclonal antibodies against β-actin (A5441, used dilution 1:10,000) and rabbit polyclonal antibodies against KTN1 or RBBP1 (Sigma-Aldrich, HPA003178 or HPA011924, used dilutions 1:500). Murine monoclonal antibody against SRP54 was purchased from BD Biosciences, Heidelberg, Germany (610940, used dilution 1:1000). Antibodies against ERj1, Sec62, and SRα were raised against the C-terminal oligopeptide of ERj1 or Sec62 plus an additional N-terminal cysteine (CELVQKKKQAKS or CGETPKSSHEKS in single letter code, used dilution 1:500) and an internal peptide of SRα plus a C-terminal cysteine (KKFEDSEKAKKPVRC, used dilution 1:500), respectively, as previously described.