α tubulin antibody

Animals were synchronized by bleaching and plated as L1 larvae on NGM agar plates seeded with E. coli HT115 (EV), which were kept at 20 °C. At the L4 larval stage, animals were transferred to plates containing 5-Fluorouracil (10 µM) (Merck, #F6627) to prevent progeny production. N2, and folr-1(syb4116) mutants were collected on day 2 of adulthood and frozen in liquid nitrogen. GMC101 and CL2122 (and crosses of these strains with folr-1(sy4116) mutant) were transferred to 25 °C on day 1 of adulthood, collected on day 2 of adulthood, and frozen in liquid nitrogen. Animals were lysed in a protease inhibitor cocktail (ThermoFisher Scientific, #78430)-supplemented urea solution (Merck, #51457) by grinding with a plastic pestle in 1.5 ml Eppendorf tubes. Lysates were resolved on 4–15% precast polyacrylamide gels (Bio-Rad, #4561083). Immun-Blot PVDF Membrane (Bio-Rad, #1620177) was used for blotting. Purified anti-β-Amyloid, 1–16 Antibody (6E10) (used with a 1:1000 dilution) was purchased from BioLegend. Anti-S6 Ribosomal Protein Antibody (used with a 1:1000 dilution) was purchased from Merck (#ZRB1172). α-tubulin antibody (used with a 1:5000 dilution) was purchased from Merck (#T5168). Clarity Western ECL Substrate (Bio-Rad, #1705061) and ChemiDoc MP-imager (Bio-Rad) were used for protein detection in Western blot.

Animals were collected at indicated age and frozen in liquid nitrogen. Animals were lysed in protease inhibitor cocktail (Abcam, #ab65621) and 3× phosphatase inhibitor (Thermo Scientific, #78420)-supplemented RIPA buffer (bioWORLD) with a micropestle in 1.5-ml Eppendorf tubes. Lysates were resolved on 4–15% precast polyacrylamide gels (Bio-Rad). Phospho-p38 MAPK (used with 1:1000 dilution) was purchased from Cell Signaling Technology (#4511S) and α-tubulin antibody (used with 1:5000 dilution) from Merck (T5168). Secondary antibodies Goat Anti-Rabbit IgG H&L (HRP) (ab97051) and Goat Anti-Mouse IgG H&L (HRP) (ab97023) (used with 1:10,000 dilution) were purchased from Abcam. SuperSignal™ West Pico PLUS Chemiluminescent Substrate (#34577, Thermo Fisher Scientific) was used for protein detection. Western blots were quantified by using Fiji (see Supplementary Table S6 for Western blot quantifications).

Cells were grown in either 1% or 10% FCS as described in the “Cell culture” section. The western blot protocol was adapted from our previous work [58 (link)]. Cells were lysed in 300 μl RIPA buffer containing protease inhibitor and phosphatase inhibitor. Fifteen to 25 μg total protein was loaded on 10% SDS PAGE, and electrophoresis was performed for 1 h at 120 V to resolve the proteins. We followed the protocol from Cell Signaling for transferring, blocking, incubating, washing, and developing the membrane. Cells treated with 1 μM thapsigargin (TG) and 1 μM hydrogen peroxide for 5 h were also included as a positive control for phosphorylation of eIf2α protein. Tubulin was used as a loading control. The following antibodies were used in the western blot analysis: Phospho-eIF2α (Ser51) Antibody (Cell Signaling #9721), eIF2 α Antibody (Cell Signaling #9722), and α-Tubulin Antibody (Merck Millipore #CP06).
For quantification, band intensities of eIF2α and p-eIF2α were normalized by the corresponding loading control (tubulin). Then, for both eIF2α and p-eIF2α, normalized band intensities were divided by the average normalized band intensity across the different conditions. Finally, the ratio of p-eIF2α to eIF2α was calculated by dividing the normalized intensities calculated in the previous step. The band intensities were quantified using the ImageJ software [60 (link)].