Tgx stain free gel

Fibroblast cells were lysed in RIPA buffer (R0278 Sigma) containing a protease inhibitor cocktail (Roche), and total protein was quantified using a Bradford Reagent protein assay (B6916 Sigma-Aldrich). Protein lysates were denatured by 1X SDS Sample Buffer. The resulting samples were incubated at 95°C for 5 min. Protein samples (100 μg) were then separated on TGX Stain-FreeTM Gels (Bio-Rad) and electroblotted onto a PVDF membrane (Trans-Blot^® TurboTM Transfer Pack/Bio-Rad). Membranes were incubated in blocking buffer (5% non-fat dried milk diluted in TBS + 0.1% Tween) for 1 h at room temperature, washed three times in TBS + 0.1% Tween for 5 min, and incubated with primary antibody (PRPF8 Abcam ab79237) at 1:500 dilution in blocking buffer overnight at 4°C. Thereafter, blots were washed three times in TBS + 0.1% Tween and incubated with secondary HRP-conjugated antibody in blocking buffer for 45 min at RT. Blots were washed another five times and protein bands were visualized using SuperSignal West Pico PLUS (Thermo Scientific) on X-ray films. β-ACTIN (monoclonal, 1:4,000,000, Sigma-Aldrich A3854) was used as a loading control. HiMark Prestained Protein Standard (LC5699, Thermo) was used as a molecular weight standard.

For SDS-electrophoresis, 4–15% precast gradient TGX Stain-Free^TM gels (Bio-Rad Laboratories, Inc.) were used and visualized by stain-free enabled imager (Gel Doc^TM EZ Imager, Bio-Rad). After blotting onto PVDF membrane (Bio-Rad), the proteins were visualized by probing with a murine horseradish peroxidase (HRP) conjugated antibody (New England BioLabs) against MBP at 1:50,000 dilution. Bands were detected by chemiluminescence using enhanced chemiluminescent (ECL) substrate (Thermo Scientific) and a digital imaging system (ImageQuant^TM LAS 4000, GE Healthcare).

Protein isolation and Western blotting were conducted essentially as described previously^{10 (link)}. 10 μg protein aliquots were electrophoresed on 4–20% TGX stain-free gels (Bio-Rad) and transferred to PVDF membranes using Trans-Blot® Turbo^TM PVDF Transfer pack (Bio-Rad). Primary antibodies and their dilutions were as specified previously^{10 (link)}, plus the following: porin (Abcam ab15895, rabbit polyclonal, 1:1,000), OXPHOS cocktail (Abcam ab110413, formerly Mitosciences ms604, mouse monoclonals for single subunits of each OXPHOS complex, namely NDUFB8, SDHB, UQCRC2, MTCOI and ATP5A, 1:250), AOX (21^st Century Biochemicals, customized rabbit polyclonal, 1:40,000), HSP60 (Abcam ab46798, rabbit polyclonal, 1:20,000), GAPDH (Cell Signaling #2118, rabbit polyclonal, 1:1,000) and LC3b (Novus NB600-1384, rabbit polyclonal, 1:1,000). Secondary antibodies and their dilutions were: goat anti-mouse IgG (Jackson ImmunoResearch #115-035-146, 1:10,000) and goat anti-rabbit IgG (Jackson ImmunoResearch #111-035-144, 1:20,000). Blot images have been cropped, rotated and framed where appropriate, and optimized for contrast and brightness, with reprobings of the same gel or different regions of the same gel shown as distinct image panels separated by white spacers. No other manipulations were introduced.

Proteins from cell lines for Western blot assays were extracted using M-PER lysis buffer (Thermo Fisher Scientific) supplemented with Halt Protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific) at 4°C for 10 minutes, and concentrations were determined by Bradford assay (Thermo Fisher Scientific). Prior to blotting, proteins were denatured in sample buffer (10% glycerol, 2% SDS, 62.5 mM Tris-HCL, pH 6.8) added with 10% β-mercaptoethanol and boiled for 5 minutes at 99°C. Western blots were performed as follows: samples were run in 4%–20% TGX stain-free gels (Bio-Rad) for 40 minutes at 180 V, and electrophoretic transfer was performed by Trans-Blot Turbo (Bio-Rad) onto PVDF membranes (Bio-Rad). Membranes were blocked in 5% Blotting-Grade Blocker for 1 hour at RT and washed 3 times in PBS-Tween (Medicago) prior to antibody staining. Antibodies used in this study are listed: anti-SOX10 1:2,000 dilution (Atlas Antibodies, catalog HPA068898), anti-MITF 1:2,000 dilution (Atlas Antibodies, catalog HPA003259), and β-actin 1:5,000 dilution (MilliporeSigma, catalog A5441).

Approximately 3E10 EVs per sample were diluted in reducing Laemmli buffer, denatured at 95 °C for 10 min, and loaded into precast 4–20% TGX Stain-free gels (Bio-Rad). Separated protein was transferred to a PVDF membrane using a Trans-Blot Turbo transfer system (Bio-Rad), and blocked in 1% casein for 1 h. Primary antibodies were diluted in blocking buffer and incubated with membranes for 1–3 h; proteins of interest were detected using HRP-conjugated secondary antibodies and chemiluminescent substrate. Primary and secondary antibody information is listed in Supplementary Table 2.

Homo sapiens Grx2, Grx5, their mutants, E. coli IscS, roGFP2, D. rerio Sirt1, and AtGrxC1 were expressed as His-tagged proteins and purified via immobilized metal affinity chromatrography^{7 (link),10 (link),66 (link)}. SDS-PAGE was performed using pre-casted TGX stain-free gels (4–20%, BioRad, Hercules CA, USA) and imaged according to the manufacturers’ instructions. Protein concentration was determined at 280 nm (ε_Grx5 = 11,585 and ε_Grx2 = 7,450 M⁻¹ cm⁻¹).