rabbit α human eif4e  (Cell Signaling Technology Inc)

Journal: Nature metabolism

Article Title: Transcriptional programming of translation by BCL6 controls skeletal muscle proteostasis

doi: 10.1038/s42255-024-00983-3

Figure Lengend Snippet: (a) Heatmap of ribosome protected fragment (RPF) sizes in sequencing libraries of tamoxifen-treated Bcl6i-MKOmRiboTag and mRiboTag mice. (b) Pie chart distribution of mapped RPF reads. (c) Empirical cumulative distribution frequency (Ecdf) plots of the translation efficiencies (natural logarithm of TE) in quadriceps for all genes. Bcl6i-MKO mRiboTag vs mRiboTag P = 2.2e-16 by two-sided Kolmogorov-Smirnov testing (d) Metagene distribution plot showing normalized ribo-seq RPF reads from the TSS to TTS for all genes (left) and genes with increased TE (right). (e) Western blot and densitometry of ubiquitin protein and actin in quadriceps from Cre− tmx and Bcl6i-MKO males 1.5 weeks after tamoxifen treatment (n = 4/group). (f) EIF4E co-immunoprecipitation and western blots for EIF4G (top) and 4EBP1 (bottom) in gastrocnemius from Cre− tmx and Bcl6i-MKO males 1 week after tamoxifen treatment (n = 2/group). (g) Western blot and densitometry of p4EBP1, Non-phospho 4EBP1, Total 4EBP1, and total (Memcode) protein in quadriceps from 10 week old Bcl6fl/fl and Bcl6MKO males (n = 4/group). P(Bcl6fl/fl vs Bcl6MKO) for p4EBP1 = 0.0009, total 4EBP1 = 0.0004, and nonP 4EBP1 = 0.0002 by unpaired, two-tailed t-test with Welch correction. (h) qPCR of protein synthesis and degradation regulators in n = 4 Cre+ oil, n = 4 Cre− tmx and n = 5 Bcl6i-MKO male mice 7 days after treatment. One-way ANOVA with Dunnett’s multiple comparisons test showed that P(Cre+ oil vs Bcl6i-MKO) for Eif4ebp1 = 0.0086, Mstn = 0.0018, Igf1 = 0.0332 and Ar = 0.0420; P(Cre− tmx vs Bcl6i-MKO) for Eif4ebp1 = 0.0163, Mstn = 0.0011, Igf1 = 0.0140 and Ar = 0.0330. (i, j) Analysis of Eif4ebp1 knockdown efficiency in muscles (n = 3 mice/group for scramble and Eif4ebp1 shRNA1 and n = 2 mice/group for Eif4ebp1 shRNA2). (i) qPCR of Eif4ebp1 and (j) western blot and densitometry of 4EBP1 in Cre− tmx and Bcl6i-MKO male mice transduced with scramble (black border), Eif4ebp1 shRNA1 (green border), or Eif4ebp1 shRNA2 (yellow border) and treated with tamoxifen one week after viral infection. Tissues were analyzed eight weeks later. All bar graph data in (e, g-j) are represented as mean ± SEM. # p < 0.05, ## p < 0.01 for Cre+ oil vs Bcl6i-MKO and * p < 0.05, ** p < 0.01, *** p < 0.001 for Cre−tmx vs Bcl6i-MKO and Bcl6fl/fl vs Bcl6MKO.

Article Snippet: The following primary antibodies were used: α-phospho-AKT Ser473 (4060), α-Pan-AKT (4691), α-LC3 A/B (12741), α-phospho-4EBP1 Thr37/Thr46 (2855), α-non-phospho4EBP1 Thr46 (4923), α−4EBP1 (9644), α-eIF4G (2469), α-phospho-S6 Ser240/Ser244 (5364), α-total S6 (2217), α-phospho-FOXO1 Ser256 (9461), α-FOXO1 (2880), α-ubiquitin (3936), α-SMAD1 (6944, Cell Signaling), α-phospho-SMAD1/SMAD5/SMAD8 (Vli31, MaineHealth Institute for Research; 1:2,000 dilution), α-Myostatin (ab98337; 1:250 dilution), AR (ab108341), VDAC (ab15895, AbCam), α-BCL6 (7388), α-eIF4E (2067, Santa Cruz Biotechnology), α-Actin (A2066), α-Tubulin (T4026; Sigma-Aldrich), α-puromycin (PMY-2A4; Developmental Studies Hybridoma Bank; 1:500 dilution) and OxPhos Rodent WB Antibody (45–8099; Thermo Fisher; 1:250 dilution).

Techniques: Sequencing, Western Blot, Ubiquitin Proteomics, Immunoprecipitation, Two Tailed Test, Knockdown, Muscles, Transduction, Infection

Journal: Nature metabolism

Article Title: Transcriptional programming of translation by BCL6 controls skeletal muscle proteostasis

doi: 10.1038/s42255-024-00983-3

Figure Lengend Snippet: a, Left: schematic of EIF4E’s interaction with 4EBP1 and EIF4G. Right: EIF4E co-immunoprecipitation and western blots for EIF4G (top), 4EBP1 (middle) and EIF4E (bottom) in quadriceps from Cre− tmx and Bcl6i-MKO males 1 week after tamoxifen treatment (n = 2 per group). b, Western blot and densitometry of p4EBP1, non-phospho-4EBP1, total 4EBP1 and actin protein in gastrocnemius from n = 3 Cre− tmx and n = 4 Bcl6i-MKO males 1 week after treatment. Cre− tmx versus Bcl6i-MKO P value for p4EBP1 = 0.0188, total 4EBP1 = 0.0319, ratio = 0.0258 and non-phospho-4EBP1 = 0.0426 by unpaired, two-tailed t-test with Welch correction. c,d, Western blot and densitometry of myostatin, AR (c), IGF-1–AKT signalling (d; pAKT, AKT, pS6, S6, pFOXO1, FOXO1) and actin or total (Licor, Memcode) protein in quadriceps from n = 3 Cre− tmx and n = 4 Bcl6i-MKO males 1 week after treatment. Cre− tmx versus Bcl6i-MKO P value for AR = 0.0133, pAKT = 0.0383, total AKT = 0.0016, pS6 = 0.048 and pFOXO1 = 0.0126 by unpaired, two-tailed t-test with Welch correction. e, Western blot and densitometry of pSMAD1/pSMAD5/pSMAD8, total SMAD1 and total (Licor) protein in quadriceps from Cre− tmx and Bcl6i-MKO males 1 week after treatment (n = 4 per group). f, Myofibre CSAs determined from skeletal muscle of Cre− tmx and Bcl6i-MKO mice infected with MyoAAV4As encoding scramble (Scrmb) or Eif4ebp1 shRNAs and then treated with tamoxifen. CSAs of 2,000 myofibres per mouse were determined 8 weeks later (n = 3 mice per group for scramble and Eif4ebp1 shRNA1 and n = 2 mice per group for Eif4ebp1 shRNA2). Whiskers in the box plot show 5–95th percentile values, the line indicates the median, and the box represents the first to third quartile values. Two-way ANOVA followed by Tukey’s multiple-comparisons test was performed. P value < 1 × 10−15 for Scrmb-Cre− tmx versus shRNA2-Cre− tmx, Scrmb-Cre− tmx versus Scrmb-Bcl6i-MKO, Scrmb-Cre− tmx versus shRNA1-Bcl6i-MKO, Scrmb-Cre− tmx versus shRNA2-Bcl6i-MKO, shRNA1-Cre− tmx versus shRNA2-Cre− tmx, shRNA1-Cre− tmx versus Scrmb-Bcl6i-MKO, shRNA1-Cre− tmx versus shRNA1-Bcl6i-MKO, shRNA1-Cre− tmx versus shRNA2-Bcl6i-MKO, shRNA2-Cre− tmx versus Scrmb-Bcl6i-MKO, shRNA2-Cre− tmx versus shRNA1-Bcl6i-MKO, shRNA2-Cre− tmx versus shRNA2-Bcl6i-MKO, Scrmb-Bcl6i-MKO versus shRNA1-Bcl6i-MKO, Scrmb-Bcl6i-MKO versus shRNA2-Bcl6i-MKO and shRNA1-Bcl6i-MKO versus shRNA2-Bcl6i-MKO. All data are represented as the mean ± s.e.m. *P < 0.05, **P < 0.01 and ****P < 0.0001.

Article Snippet: The following primary antibodies were used: α-phospho-AKT Ser473 (4060), α-Pan-AKT (4691), α-LC3 A/B (12741), α-phospho-4EBP1 Thr37/Thr46 (2855), α-non-phospho4EBP1 Thr46 (4923), α−4EBP1 (9644), α-eIF4G (2469), α-phospho-S6 Ser240/Ser244 (5364), α-total S6 (2217), α-phospho-FOXO1 Ser256 (9461), α-FOXO1 (2880), α-ubiquitin (3936), α-SMAD1 (6944, Cell Signaling), α-phospho-SMAD1/SMAD5/SMAD8 (Vli31, MaineHealth Institute for Research; 1:2,000 dilution), α-Myostatin (ab98337; 1:250 dilution), AR (ab108341), VDAC (ab15895, AbCam), α-BCL6 (7388), α-eIF4E (2067, Santa Cruz Biotechnology), α-Actin (A2066), α-Tubulin (T4026; Sigma-Aldrich), α-puromycin (PMY-2A4; Developmental Studies Hybridoma Bank; 1:500 dilution) and OxPhos Rodent WB Antibody (45–8099; Thermo Fisher; 1:250 dilution).

Techniques: Immunoprecipitation, Western Blot, Two Tailed Test, Infection