Journal: bioRxiv

Article Title: Active role of free RNA during the early phase of proteostasis stress

doi: 10.1101/564799

Figure Lengend Snippet: (A) Enrichment values of the subunits of eIF2 in individual experiments (black symbols) and the enrichment averages (red symbols). (B) Left panels, amounts of total eIF2α and its serine 51-phosporylated form (p-eIF2α) as determined by western blotting. Right panels, enrichment of non-phosphorylated eIF2α on RNA upon heat shock. *p<0.05, two-tailed t-test; N=3 independent experiments (mean + SD). (C) Enrichment values of the subunits of the m A methyltransferase TRMT6/61A in individual experiments (black symbols) and the enrichment averages (red symbols). (D) Upper panel, amounts of TRMT6 as determined by western blotting. Lower panel, enrichment of TRMT6 on RNA upon heat shock. ***p<0.001, two-tailed t-test; N=3 independent experiments (mean + SD). (E) Increased association of TRMT6 with mRNAs during heat shock as determined by western blot analysis of PolyT pulldowns. ***p<0.001, two-tailed t-test, N=3 independent experiments (mean + SD).

Article Snippet: The following antibodies were used: anti-TRMT6 (A303-008A-M) from Bethyl (Montgomery, TX); anti-TRMT61A (sc-107105) and anti-S6 (sc-74459) from Santa Cruz Biotechnology (Dallas, TX); anti-eIF2α (9722), anti-eIF2α (phosphor-Ser51) (3398), anti-eIF4E (9742), HRP-conjugated anti-rabbit-IgG (7074) and Alexa Fluor 647-conjugated anti-rabbit IgG (4414) from Cell Signaling (Danvers, MA); anti-Flag (F1804) and HRP-conjugated anti-mouse IgG (A9044) from Sigma-Aldrich.

Techniques: Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Active role of free RNA during the early phase of proteostasis stress

doi: 10.1101/564799

Figure Lengend Snippet: (A) Increased association of eIF2α with TRMT61A-containing complexes during heat shock as determined by immunoprecipitation (IP) of Flag-tagged TRMT6/61A. *p<0.05, two-tailed t-test; N=3 independent experiments (mean + SD). (B) RNA hydrolysis (+nuclease) abolishes association of eIF2α with the TRMT61A-containing complexes. One representative out of three independent experiments is shown.

Article Snippet: The following antibodies were used: anti-TRMT6 (A303-008A-M) from Bethyl (Montgomery, TX); anti-TRMT61A (sc-107105) and anti-S6 (sc-74459) from Santa Cruz Biotechnology (Dallas, TX); anti-eIF2α (9722), anti-eIF2α (phosphor-Ser51) (3398), anti-eIF4E (9742), HRP-conjugated anti-rabbit-IgG (7074) and Alexa Fluor 647-conjugated anti-rabbit IgG (4414) from Cell Signaling (Danvers, MA); anti-Flag (F1804) and HRP-conjugated anti-mouse IgG (A9044) from Sigma-Aldrich.

Techniques: Immunoprecipitation, Two Tailed Test

Journal: Redox Biology

Article Title: Polysulfide and persulfide-mediated activation of the PERK-eIF2α-ATF4 pathway increases Sestrin2 expression and reduces methylglyoxal toxicity

doi: 10.1016/j.redox.2024.103450

Figure Lengend Snippet: Polysulfide-induced phosphorylation of eIF2α and nuclear accumulation of ATF4 are mediated by PERK phosphorylation in SH-SY5Y cells. (A) Cells were treated with 100 μM Na 2 S 2 for the indicated time, and levels of p-eIF2α and eIF2α in the cytoplasmic fractions were analyzed by western blotting. (B) Cells were treated with the indicated Na 2 S 2 concentrations for 2 h, and levels of p-eIF2α and eIF2α in cytosolic fractions were analyzed by western blotting. The p-eIF2α band densities were measured, and the ratio to eIF2α was calculated and expressed as the fold-change of the band levels relative to 0 h (A) or 0 μM (B) samples. (C) Cells were treated with 100 μM Na 2 S 2 for the indicated time, and ATF4 levels in the nuclear fractions were analyzed by western blotting. LaminB1 was used as a nuclear loading control (bottom panel). (D) Cells were treated with the indicated Na 2 S 2 concentrations for 2 h, and ATF4 levels in the nuclear fractions were analyzed by western blotting. LaminB1 was used as a nuclear loading control (bottom panel). The ATF4 band densities were measured, and the ratio to LaminB1 was calculated and expressed as the fold-change of the band levels relative to the 0 h (C) or 0 μM (D) samples. (E and F) SH-SY5Y cells transfected with siRNA were treated with 100 μM Na 2 S 2 for 0.5 h (for cytoplasmic proteins) or 2 h (for nuclear proteins) and lysed to prepare the cytoplasmic and nuclear fractions, respectively. Western blotting was performed on the cytoplasmic or nuclear proteins using anti-PERK (E) or anti-ATF4 antibodies, respectively (F). β-actin and LaminB1 were used as loading controls for cytoplasmic and nuclear proteins, respectively. Protein levels were quantified by densitometry, normalized to the loading control (β-actin or LaminB1), and expressed as a fold of the protein level detected in si Ctrl-transfected (scrambled) cells. Values indicate means ± S.D. Dunnett's test (A–D). Tukey's test (E and F). ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. All data are representative of at least three independent experiments.

Article Snippet: The membranes were incubated with the following primary antibodies: ATF4 (10835-1-AP, Proteintech), CBS (ab96252, Abcam), eIF2α (GTX101241, Gene Tex), p-eIF2α (Ser51) (3398, Cell Signaling Technology), 3-MST (HPA001240, Sigma-Aldrich), Nrf2 (H-300; Santa Cruz Biotechnology, CA, USA), PERK (3192, Cell Signaling Technology), p-PERK (ab192591 Phospho T982, Abcam), PTP1B (5311, Cell Signaling Technology), Sestrin2 (10795-1-AP, Proteintech), β-actin (5125, Cell Signaling Technology), LaminB1 (12586, Cell Signaling Technology), and β-Tubulin (5346, Cell Signaling Technology).

Techniques: Western Blot, Control, Transfection