rabbit anti-eif4e monoclonal antibody  (Cell Signaling Technology Inc)

Journal: iScience

Article Title: The PRMT5-splicing axis is a critical oncogenic vulnerability that regulates detained intron splicing

doi: 10.1016/j.isci.2025.112965

Figure Lengend Snippet: HCT116 cells are CLNS1A-independent until PRMT5 activity is decreased (A) Western blot analyses of HA (CLNSIA), SDMA (methyl-SmB), and HSP90 as loading control in HCT116 dTag-CLNS1A #1 ( Left ) and #2 ( Right ) maintained in 1 μM dTag-13 for the indicated number of days. (B) Representative Western blot analysis of CLNS1A, SDMA (methyl-SmB), and loading control GAPDH levels in HCT116 sgCtrl and four sgCLNS1A clones ( n = 4 biological replicates). (C) Relative proliferation of HCT116 sgCtrl and four sgCLNS1A clones through analyses of cumulative population doubling over a 6-day period. Data represent mean ± SD of 10 technical replicates. (D) Relative viability of HCT116 sgCtrl and indicated sgCLNS1A clones treated with the PRMT5i JNJ-64619178 for 6 days. Data represent mean ± SD of 3 technical replicates. (E–G) HCT116 parental or two dTag-CLNS1A clones treated with or without 1μM dTag-13 in combination with the indicated concentrations of JNJ-64619178 (PRMT5i). (E) Relative viability was assessed after treatment for 6 days. Data represent mean ± SD of 3 technical replicates. Red markings correspond to the critical PRMT5i doses used in F and 5G. (F) Western blot analyses of HA (CLNS1A), SDMA, and loading control HSP90 after treatment with the critical indicated PRMT5i doses for 3 days (G) Relative levels of a representative DI in EIF4E were assessed by qRT-PCR after treatment with the critical indicated PRMT5i doses for 3 days. Data represent mean ± SD of 3 technical replicates. For all panels, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001, Student’s t test.

Article Snippet: Rabbit anti-eIF4E Monoclonal Antibody , Cell Signaling Technology , Cat#2067; RRID: AB_2097675.

Techniques: Activity Assay, Western Blot, Control, Clone Assay, Quantitative RT-PCR

Journal: Cells

Article Title: Calcitriol Suppresses HIF-1 and HIF-2 Transcriptional Activity by Reducing HIF-1/2α Protein Levels via a VDR-Independent Mechanism

doi: 10.3390/cells9112440

Figure Lengend Snippet: Calcitriol suppresses HIF-1/2α subunits protein synthesis at the level of HIF1A and EPAS1 mRNA translation by downregulating components of the PI3K/Akt/mTOR signaling pathway. Western blot analysis of ( A ) phosphorylated Akt and total Akt (numbers below blots represent P-AKT/AKT ratios from three independent experiments. In each case, ratio value of 1 is given to normoxic conditions in the absence of calcitriol). ( B ) phosphorylated eIF4E and phosphorylated 4E-BP1 protein levels in Huh7 cell lysates. Cells were treated with 1,25(OH) 2 D3 (100 nM) and incubated under hypoxia (1% O 2 ) for 8 or 24 h as indicated. Actin is used as a loading control for A. Tubulin is used as a loading control for B.

Article Snippet: Proteins were resolved by 10% SDS-PAGE gels and analyzed by Western immunoblotting using the antibodies: rabbit polyclonal antibody against HIF-2α (ΝΒ100-122, 1:750 dilution; Novus Europe, Cambridge, UK), affinity purified rabbit polyclonal antibody against HIF-1α (1:1000 dilution, reported in [ ]), mouse monoclonal antibody against ARNT (611079, 1:500; BD Biosciences, San Jose, CA, USA), mouse monoclonal antibody against β-actin (3700, 1:5000 dilution; Cell Signaling, Danvers, MA, USA), rabbit monoclonal antibody against phospho-Akt (Ser473) (4060, 1:1000 dilution; Cell Signaling), rabbit monoclonal antibody against Akt (pan) (4685, 1:1000 dilution; Cell Signaling), rabbit polyclonal antibodies against ERK1/2 and Phospho-ERK1/2 (9102 and 9101, respectively, 1:1000 dilution; Cell Signaling), rabbit monoclonal antibody against phospho-4E-BP1 (Thr37/46) (236B4) (2855, 1:1000 dilution; Cell Signaling), rabbit monoclonal antibody against phospho-eIF4E (Ser209) antibody (9741, 1:1000 dilution; Cell Signaling) mouse polyclonal antibody against VDR (H00007421-B01P, 1:500 dilution; Abnova), mouse monoclonal antibody against α-Tubulin (DM1A) (3873, 1:10000 dilution; Cell Signaling) and a rabbit polyclonal antibody against VEGF (sc152, 1:250; Santa Cruz Biotechnology, TX, USA).

Techniques: Western Blot, Incubation, Control

Journal: Cells

Article Title: Calcitriol Suppresses HIF-1 and HIF-2 Transcriptional Activity by Reducing HIF-1/2α Protein Levels via a VDR-Independent Mechanism

doi: 10.3390/cells9112440

Figure Lengend Snippet: Calcitriol suppresses HIF-1/2α subunits protein synthesis at the level of HIF1A and EPAS1 mRNA translation by downregulating components of the PI3K/Akt/mTOR signaling pathway. Western blot analysis of ( A ) phosphorylated Akt and total Akt (numbers below blots represent P-AKT/AKT ratios from three independent experiments. In each case, ratio value of 1 is given to normoxic conditions in the absence of calcitriol). ( B ) phosphorylated eIF4E and phosphorylated 4E-BP1 protein levels in Huh7 cell lysates. Cells were treated with 1,25(OH) 2 D3 (100 nM) and incubated under hypoxia (1% O 2 ) for 8 or 24 h as indicated. Actin is used as a loading control for A. Tubulin is used as a loading control for B.

Article Snippet: Proteins were resolved by 10% SDS-PAGE gels and analyzed by Western immunoblotting using the antibodies: rabbit polyclonal antibody against HIF-2α (ΝΒ100-122, 1:750 dilution; Novus Europe, Cambridge, UK), affinity purified rabbit polyclonal antibody against HIF-1α (1:1000 dilution, reported in [ ]), mouse monoclonal antibody against ARNT (611079, 1:500; BD Biosciences, San Jose, CA, USA), mouse monoclonal antibody against β-actin (3700, 1:5000 dilution; Cell Signaling, Danvers, MA, USA), rabbit monoclonal antibody against phospho-Akt (Ser473) (4060, 1:1000 dilution; Cell Signaling), rabbit monoclonal antibody against Akt (pan) (4685, 1:1000 dilution; Cell Signaling), rabbit polyclonal antibodies against ERK1/2 and Phospho-ERK1/2 (9102 and 9101, respectively, 1:1000 dilution; Cell Signaling), rabbit monoclonal antibody against phospho-4E-BP1 (Thr37/46) (236B4) (2855, 1:1000 dilution; Cell Signaling), rabbit monoclonal antibody against phospho-eIF4E (Ser209) antibody (9741, 1:1000 dilution; Cell Signaling) mouse polyclonal antibody against VDR (H00007421-B01P, 1:500 dilution; Abnova), mouse monoclonal antibody against α-Tubulin (DM1A) (3873, 1:10000 dilution; Cell Signaling) and a rabbit polyclonal antibody against VEGF (sc152, 1:250; Santa Cruz Biotechnology, TX, USA).

Techniques: Western Blot, Incubation, Control