Mini protean precast mini page gels

Total protein concentrations of lung homogenate lysates were analyzed with a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). SDS-PAGE was performed using Mini-PROTEAN^® Precast Mini PAGE Gels (Bio-Rad, Hercules, CA). Trans-Blot Turbo Mini 0.2 µM PVDF Transfer Packs (Bio-Rad) were used for transferring of proteins to the PVDF membranes. Membranes were blocked for 3 h at RT and incubated with primary antibodies (rabbit anti-mouse NF-κB(RelA/p65/) (sc-8008; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-mouse phospho (p)-RelA/p65(NF-κB) (Ser276; A1953; Abcam), mouse anti-mouse arginase-1 (ab239731), rabbit anti-mouse CD206 (ab64693) and rabbit anti-mouse GAPDH (14C10; Cell Signaling; 1:500) in blocking buffer. After washing with PBS-Tween, membranes were incubated with secondary antibodies (Alexa Fluor 488-conjugated goat anti rabbit/mouse (Invitrogen, Carlsbad, CA)) for 1 h RT. Imaging of blots was preformed using a ChemiDoc system (Bio-Rad) followed by quantification with densitometry normalized to GAPDH.

Total protein concentrations of lung homogenate lysates were determined and SDS-PAGE was performed using Mini-PROTEAN^® Precast Mini PAGE Gels (Bio-Rad, Hercules, CA). Trans-Blot Turbo Mini 0.2 µM PVDF Transfer Packs (Bio-Rad) were used for transferring of proteins to the PVDF membranes. Membranes were blocked for 3h at RT and incubated with primary antibodies (rabbit anti-human TRAP5b (provided by Dr. Göran Andersson), rabbit polyclonal to TRAP5 (Cat #PA5-116970; Invitrogen, MA, USA), and rabbit anti-mouse GAPDH (Cat #MA5-15738-D680; Invitrogen 1:500) in blocking buffer. After washing with PBS-Tween, membranes were incubated with secondary antibodies (Alexa Fluor 488-conjugated goat anti rabbit/mouse (Invitrogen, Carlsbad, CA) for 1h RT. Imaging of blots was preformed using a ChemiDoc system (Bio-Rad) followed by quantification with densitometry normalized to GAPDH.

To assess the levels of SpmX in different strains (fig. S2H), cells were grown in M2 minimal media supplemented with 1 mM glucose at 30°C. Cells were incubated with or without indicated concentrations of xylose for 30 min to enable protein induction. One milliliter of cells was harvested at an OD₆₀₀ of 0.35 to 0.4. Harvested cells were normalized to the lowest OD₆₀₀ of the collected cells, pelleted by centrifugation at 20,000g for 2 min, and resuspended in 30 μl of 2× sample buffer [20 mM tris-HCl (pH 7.0), 6% SDS, 10 mM EDTA, 20% glycerol (v/v), and 10% β-mercaptoethanol (v/v)] and incubated at 98°C for 10 min. Twenty microliters of this sample was loaded on Mini-PROTEAN Precast Mini PAGE Gels (Bio-Rad) for separation. Proteins separated via SDS-PAGE were transferred using a semidry platform to PVDF membranes at 80 V for 1 hour at room temperature. SpmX levels on the PVDF membrane were detected using a chemiluminescent substrate (SuperSignal West PICO PLUS, Thermo Pierce) after incubation with an anti-SpmX (1:10,000) primary antibody raised against the SpmX-IDR (7 (link)) and a polyclonal goat anti-mouse HRP (1:10,000)–conjugated secondary antibody (Abcam). SpmX levels were estimated using Fiji from three independent biological replicates.