Supersignalh west pico chemiluminescent substrate

Following our previous protocol (Ding et al., 2011 (link)) with modification, larval zebrafish proteins were extracted with lysis buffer and separated using 12% SDS-polyacrylamide gel electrophoresis. The protein bands were transferred onto a nitrocellulose membrane and blocked the membrane with TBS containing 5% skim milk. The membranes were incubated either with mouse anti-LC3 antibody (M186-3; MBL Biotechnology, Inc.) at 1:1,000 dilution or with rabbit anti-P62 antibody (PM 045; MBL Biotechnology, Inc.) at 1:2,000 dilution in TBS containing 1% skim milk; then the membrane was washed and incubated with secondary antibodies (HRP-conjugated goat anti-mouse or goat anti-rabbit IgGs; both 1:2,000 dilutions; Zhongshanjinqiao Co. China) for 2 h at RT. Chemiluminescent signals were detected using the Supersignal H West Pico chemiluminescent substrate (Thermo) with a GE Imaging System (GE Corporation).

For western immunoblotting, cell lysates of exponentially growing LB cultures were obtained by centrifuging cells corresponding to an OD600 = 10 and resuspending the cell pellet in Laemmli buffer^{45 (link)}. Prior to protein separation by SDS/PAGE using 12% (wt · vol⁻¹) polyacrylamide gels the samples were heated to 95 °C for 5 min Subsequently, the proteins were transferred to a nitrocellulose Roti-PVDF membrane (Roth) by semidry transfer. The polar flagellins FlaA and FlaB (of S. putrefaciens and S. oneidensis alike) were detected with polyclonal antibodies which were raised against the N-terminal conserved region of S. MR-1 FlaB (Eurogentec Deutschland) in the dilution of 1:500. As secondary antibody anti-rabbit IgG-horseradish peroxidase (Thermo Fisher Scientific, prod. # 31460) was used at a dilution of 1:20,000. FLAG-tagged FliS was detected with a monoclonal, horseradish-peroxidase-conjugated antibody raised against the FLAG-tag (Sigma Aldrich, prod. # A8592) in the dilution of 1:1000. The horseradish peroxidase signal was detected with the CCD System LAS 4000 (Fujifilm) after incubating the membranes with SuperSignalH West Pico Chemiluminescent Substrate (Thermo Scientific) for one minute. If portions of gels or blots are shown the full gels or blots can be found in Supplementary Figure 12.