Low fluorescence pvdf membrane

Due to variability of the villin promoter on expression [35 (link)], hTHTR2 protein expression was evaluated in various tissues. Tissue extracts were prepared in RIPA lysis buffer (Thermo Fisher, Waltham, MA, USA) with dissolved Pierce protease inhibitor tablets (Thermo Fisher) followed by homogenization. After centrifuging the homogenates at 15,000× g and at 4 °C for 15 min, supernatants were collected, and the protein content was determined by Pierce BCA Protein Assay Kit (Thermo Fisher). Samples of 20 μg of protein were electrophoresed on precast Mini-protean TGX gels with 4–20% polyacrylamide (Bio-Rad, Hercules, CA, USA), transferred to a low-fluorescence PVDF membranes (Thermo Scientific), and then blocked in Tris-buffered saline with 0.1% Tween 20 (TBS-T) supplemented with 5% non-fat dry milk at room temperature for 1 h. Membranes were incubated overnight at 4 °C with the 1:1000 dilutions of rabbit anti-SLC19A3 primary antibodies (1:1000, Sigma-Aldrich, St. Louis, MO, USA) in TBS-T supplemented with 5% non-fat dry milk, then washed with TBS-T and incubated with 1:2000 dilutions of secondary horse radish peroxidase conjugated goat anti-rabbit IgG (Cell Signaling Technologies, Danvers, MA, USA). Signals were detected using the enhanced chemiluminescence SuperSignal West Femto Maximum Sensitivity Substrate detection kit (Thermo Fisher).

Protein samples (25 μg) were denatured with Laemmli buffer and heated at 95 °C for 10 min. The separation was performed by SDS-PAGE in 12.5% gels. Proteins were then transferred onto low fluorescence PVDF membranes (Thermo Fisher Scientific) by electroblotting at 300 mA for 1 h. After blocking overnight in 5% milk/TBS/0.05% Tween-20 (TBST), membranes were washed and probed with primary monoclonal antibody (POM1, Prionics; GRP78/HSP5, Thermo Fisher Scientific) in 1% milk/TBST for 1 h at RT. After three washes with TBST, membranes were probed with secondary antibody goat anti-mouse IgG conjugated to AF488 (Invitrogen). Subsequently, the membranes were thoroughly washed with TBST and allowed to dry before fluorescence imaging using a Typhoon Trio scanner (GE Healthcare). The membranes were reprobed with anti β-actin antibody for protein load verification, either directly conjugated to AF647 (Santa Cruz) or indirectly using a goat anti-mouse IgG conjugated to AF647 (Invitrogen).

Cells were harvested in lysis buffer (50 mM Tris, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 0.1% sodium dodecyl sulfate (SDS), 0.1% Triton-X-100, and 0.5% sodium deoxycholate) containing protease and phosphatase inhibitors (Halt protease inhibitor cocktail and phosphatase inhibitor cocktail, Thermo Fisher). Protein concentration was determined by BCA assay (Thermo Fisher). Uniform amounts of protein per sample were run on 10 or 15% polyacrylamide gels (Bio-Rad) and transferred to Immobilon-FL PVDF membranes (MilliporeSigma). For analysis of granulins, samples were transferred onto low-fluorescence PVDF membranes with 0.2 μm pores (Thermo Fisher). Membranes were blocked with protein-free blocking buffer (Thermo Fisher), then probed overnight with primary antibodies. The following day, membranes were probed with species-matched IRdye-conjugated secondary antibodies (Li-COR Biosciences) and scanned on an Odyssey scanner (Li-COR Biosciences). Blots of cell lysates were probed for Gapdh to confirm equal protein loading. Blots of conditioned media were conducted volumetrically (equivalent amounts of media loaded per lane). As neurons were plated at equal density and we detected no significant differences in cell number (Fig. 5), no additional loading control was analyzed for blots of conditioned media.