Lumino image analyzer las 4000 mini

The in vivo proteolytic activity of AaRseP was analyzed using E. coli KK211 (ΔrseA, ΔrseP) cells (Kanehara et al., 2002) as described previously (Akiyama et al., 2015 , Hizukuri et al., 2017) . E. coli KK211 (ΔrseA, ΔrseP) cells harboring pYH124 (HA-MBP-RseA(LY1)148) were transformed with a plasmid pKK11 (EcRseP-His6-Myc) (Kanehara et al., 2001) , pTM748 (AaRseP-His8), pTWV228 or plasmids encoding their derivatives.
M9 medium (without CaCl2) (Miller, 1972) supplemented with 20 µg/mL of each of the 20 amino acids, 2 µg/mL thiamine, 0.4% glucose, 1 mM IPTG and 5 mM cAMP was inoculated with transformed E. coli KK211 cells and grown at 30°C for 3 hours. Proteins were precipitated by trichloroacetic acid (TCA) treatment and separated by Laemmli SDS-PAGE. Immunoblots with anti-HA, anti-His or anti-SecB antibodies were visualized by Lumino image analyzer LAS-4000 mini (Cytiva, Tokyo, Japan (GE Healthcare)) using ECL Prime Western Blotting Detection Reagents (Cytiva, Tokyo, Japan (GE Healthcare)).
Rabbit polyclonal anti-HA (HA-probe (Y-11), Santa Cruz Biotechnology) and anti-SecB (a gift from Shoji Mizushima's Lab.) antibodies were used for immunoblotting. For detection of His-tagged proteins, anti-His antibodies from the Penta-His HRP Conjugate Kit (Qiagen) were used.