Anti-PKR (phospho T446) antibody [E120]

• IP

Lab

Immunoprecipitation - Anti-PKR (phospho T446) antibody [E120] (AB32036)

PKR was immunoprecipitated from 0.35 mg HeLa (Human cervix adenocarcinoma epithelial cell) treated with Calyculin A(100nM 30min) whole cell lysate 10 μg with ab32036 at 1/50 dilution (2μg). VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) treated with Calyculin A(100nM 30min) whole cell lysate 10 μg

Lane 2 : ab32036 IP in HeLa treated with Calyculin A(100nM 30min) whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab32036 in HeLa treated with Calyculin A(100nM 30min) whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Lower bands could be cleavage form. (PMID : 28702377)

All lanes:

Immunoprecipitation - Anti-PKR (phospho T446) antibody [E120] (ab32036)

Predicted band size: 62 kDa

Observed band size: 68 kDa

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• WB

Unknown

Western blot - Anti-PKR (phospho T446) antibody [E120] (AB32036)

All lanes:

Western blot - Anti-PKR (phospho T446) antibody [E120] (ab32036) at 1/1000 dilution

Lane 1:

Hela (human epithelial cell line from cervix adenocarcinoma) cell lysate

Lane 2:

Hela cell lysate + IFN + CalyculinA

Lane 3:

Hela cell lysate + Pptase

Predicted band size: 62 kDa

Observed band size: 68 kDa

false

• WB

Lab

Western blot - Anti-PKR (phospho T446) antibody [E120] (AB32036)

Blocking/Diluting buffer : 5% NFDM/TBST

Positive control : Rabbit monoclonal [Y117] to PKR (ab32506).

All lanes:

Western blot - Anti-PKR (phospho T446) antibody [E120] (ab32036) at 1/2000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma) whole cell lysate at 15 µg

Lane 2:

HeLa (human cervix adenocarcinoma) treated with 100 ng/mL Calyculin A whole cell lysate at 15 µg

Lane 3:

HeLa (human cervix adenocarcinoma) treated with 100 ng/mL Calyculin A followed by 20 ng/mL TNF alpha whole cell lysate at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 62 kDa

Observed band size: 68 kDa

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Exposure time: 10s

• WB

CiteAb

Western blot - Anti-PKR (phospho T446) antibody [E120] (AB32036)

Western Blotting using Anti-PKR (phospho T446) antibody [E120], ab32036. Publication image from Lee, S. et al., 2020, Genome Biol, 32039742. Legend direct from paper.

Inflammatory response mRNAs are selectively translated in G0 leukemic cells, where canonical translation is inhibited. a Repression of canonical translation. b Polysome profiles of S+, SS, and AraCS are shown. Polysome-associated mRNAs were isolated and analyzed by microarray. Graph of polysome to monosome (P/M) ratios in S+, SS, and AraCS. c, d Western analysis of translation initiation regulators : c eIF2α, phospho-eIF2α, and its regulators phospho-PERK and phospho-PKR, and d of translation regulator, eIF4EBP (4EBP) at Thr37/40 and Ser65 phosphorylation sites and total levels with quantification below. e Number of differentially expressed genes. f Venn diagram of transcriptionally and translationally upregulated genes in G0 cells induced by AraC and SS, compared to S+ cells, is shown on the left. Heatmap of gene expression changes at the transcriptome, translatome, and ribosome occupancy (RO) levels is shown on the right. See also Additional file 2 : Table S1 for the 490 translationally upregulated genes and their RO changes. g Gene ontology (GO) analyses of differentially expressed genes shown in Fig. 2e. Statistical significance of enriched GO categories is shown as a heatmap. h Expression of signature genes of G0 leukemic cells in published transcriptomes of in vivo resistant leukemic and G0 models. i Translatome analysis of G0 cells from five different cell types. Heatmap of normalized enrichment score (NES) is shown. *p ≤ 0.05. Data are represented as average ± SEM. See also Additional file 1 : Figures S1, S2 and Additional file 2 : Table S1

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