Ponceau stain

2 million macrophages were lysed in 200 μL of RIPA buffer (Tris-HCl, pH 7.4, 50 mM, NaCl 150mM, NP-40 1%, sodium deoxycholate 0.5%, SDS 0.1%) with a cocktail of protease and phosphatase inhibitors (Roche). 20 μg of protein was loaded per lane and separated on a 4%–12% SDS/PAGE gel and transferred to PVDF. Ponceau stain (Sigma) determined protein transfer and membrane was blocked for 1 h with 5% skim milk in TBS/0.1% Tween at 25°C, and then incubated with the indicated primary antibody in 3% BSA TBS/0.1% Tween overnight at 4°C. Specific antibodies Influenza NP protein (A. Sastre-Garcia) were used to quantify virus protein in the lung tissue. β-actin (Sigma) was used as a loading control. Anti-pan-AGO for immunoblotting (7G1.1) was from the Darnell laboratory (Rockefeller University), anti-AGO1 antibody was from Abcam.

For Western blot analysis Lcells, NCad-Venus Lcells and hippocampal neurons grown in 6 cm cell culture dishes were washed with PBS and lysed in 1.5 x LDS sample buffer (life technologies) supplemented with DTT (10 x reducing agent, life technologies) and protease inhibitors (Calbiochem). Lysate aliquots equivalent to 5 μg of total protein were loaded on 4 – 12 % BisTris NuPAGE gels (life technologies), separated by electrophoresis with MOPS running buffer (life technologies) and transferred to Immobilon-FL (Millipore) using the TurboBlot system (Biorad) at 25 V, 2.5 A for 7 min. Ponceau stain (Sigma) was used to validate homogeneous loading and transfer. Membranes were blocked for 1 h with Odyssey blocking buffer (Li-cor) and incubated overnight at 4 °C with primary antibodies in a 1 + 1 mixture of Odyssey blocking buffer and PBS containing 0.2 % Tween20. Membranes were rinsed several times with water followed by 2 × 10 min washes in PBS containing 0.2 % Tween20. Secondary antibodies coupled to IRdye680 and IRdye800 (Li-cor, 1:15.000) were applied for 1 h in a 1 + 1 mixture of Odyssey blocking buffer and PBS containing 0.2 % Tween20. Membranes were washed for 10 min in PBS containing 0.2 % Tween20 and 0.01 % SDS, 10 min in PBS containing 0.2 % Tween20 and rinsed several times in water before the signal was scanned using an Odyssey Infrared imager (Li-cor).

J774 cells were treated as indicated and lysates and supernatants were prepared as previously described and separated by SDS-PAGE. Proteins within the gel were transferred onto nitrocellulose and washed with TBS for 5 minutes. The membrane was then placed in Ponceau Stain (0.1% Ponceau S (Sigma-Aldrich) in 5% acetic acid) for 5 minutes, and washed with water tree times for 5 minutes each.

SUnSET was previously used to measure levels of general protein translation in E. histolytica (Hendrick et al., 2016 (link)). Wildtype trophozoites were exposed to vehicle control, 10 mM DTT, 1 mM SNP, 300 μM DPTA-NONOate, or 350 μM BFA for 1 h at 37°C. Then, to assess levels of general protein levels in stressed and unstressed cells, we incubated cells (2×10⁵) with 10 μg mL⁻¹ puromycin (Sigma-Aldrich) for 15 min before or after incubation with 100 μg mL⁻¹ cycloheximide for 10 min. All incubations were held at 37°C. Next, cells were pelleted, and proteins were precipitated using 20% (v/v) TCA and incubating on ice for 10 min. Proteins were separate by centrifugation at 2200 × g for 5 min (4°C) and washed with 5% (v/v) TCA. The protein pellet was resuspended in 2X SDS running buffer and incubated at 100°C for 10 min. The lysates were immediately analyzed using western blotting as described above. Primary mouse anti-puromycin monoclonal antibodies (Sigma-Aldrich) were used at a dilution of 1:100 and secondary horseradish peroxidase-conjugated goat anti-rabbit antibodies (Fisher Scientific) were used at a dilution of 1:2500. As a loading control, matching PVDF membranes were stained with Bio-Safe Coomassie G-250 Stain (Bio-Rad Laboratories) or Ponceau Stain (Sigma-Aldrich).