Can get signal solution

sodiumdodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 10% or 12% polyacrylamide gels and matrix-assisted laser desorption/ionization time-of-flight mass analyses were performed as described elsewhere (Hishinuma et al. 2008 (link)). The amount of cells (OD₆₀₀) was defined as the product of the volume (mL) and the optical density (600 nm) of the culture used.
For western blot analysis, protein samples prepared from cell lysates of P. putida were separated by SDS-PAGE and then transferred to PVDF membranes with an iBlot Dry blotting system (Invitrogen, Carlsbad, CA),and membranes are processed in Can Get Signal solutions (Toyobo, Osaka, Japan) in accordance with the manufacturers’ protocols. Immunological detections of P. putida σ³² and DnaJ were performed using a rabbit polyclonal antibody prepared against S. marcescens σ³² (Abcam, Cambridge, MA), and an Hsp40 antibody prepared against E. coli DnaJ (Abcam), respectively, and visualized with an alkaline phosphatase-conjugated goat anti-rabbit IgG. Detection of alkaline phosphatase activity was carried out in accordance with the instructions on the DIG detection kit (Roche Diagnostics).

Kidneys were fixed in 4% buffered paraformaldehyde and embedded in paraffin. Paraffin sections (2 μm) were stained with periodic acid-Schiff (PAS) or used for immunostaining. Information about the primary antibodies used in the study, the antigen retrieval method, and dilution ratios are listed in S1 Table. Antigen retrieval was performed by heating in citrate buffer (0.1 mol/L, pH 6.0) with a microwave or autoclave. CanGet signal solutions (Toyobo, Osaka, Japan) were used for Dach1 and WT1. For double immunostaining, the signal of the first antibody (anti-synaptopodin) was visualized by diaminobenzidine (DAB), and the slides were washed in 0.1 M glycine buffer (pH 2.2) for 30 minutes, 3 times. Then, the slides were stained with the second antibody (Dach1 or WT1) and visualized by Ni, Co-DAB. Information regarding antibodies is shown in S1 Table.

The antigen of antibody 0614 was confirmed by Western blotting. Recombinant CD73 with a C-terminal His-tag was loaded onto SDS-PAGE gel, separated, and then transferred to PVDF membranes (Bio-Rad). Membranes were blocked for 5 min with Bullet Blocking One for Western blotting (Nacalai Tesque). The membranes were washed three times with TBS-T. The blots were then incubated with antibody 0614 (10 µg/mL) or anti-His tag antibody (1:10,000, ProteinTech, Rosemont, IL, USA) at room temperature for 1 h. The membranes were washed three times with TBS-T and then incubated with HRP-conjugated anti-rat IgG (1:5000; Jackson Immuno Research, West Grove, PA, USA) or anti-mouse IgG (1:5000; CST, Danvers, MA, USA) at room temperature for 1 h. Can Get Signal solution (Toyobo, Osaka, Japan) was used to reduce background noise. After the membranes were washed three times with TBS-T, proteins were visualized using ECL prime (GE Healthcare).