Anti-METTL1 antibody

• WB

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Western blot - Anti-METTL1 antibody (AB157097)

All lanes:

Western blot - Anti-METTL1 antibody (ab157097) at 0.4 µg/mL

Lane 1:

293T whole cell lysate at 50 µg

Lane 2:

HeLa whole cell lysate at 50 µg

Lane 3:

Jurkat whole cell lysate at 50 µg

Lane 4:

TCMK-1 whole cell lysate at 50 µg

Predicted band size: 31 kDa

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Exposure time: 3min

• WB

CiteAb

Western blot - Anti-METTL1 antibody (AB157097)

Western Blotting using Anti-METTL1 antibody, ab157097. Publication image from Balasubramanian, S. et al., 2019, Mol Cell, 31031083. Legend direct from paper.

m7G Position Is Essential for let-7e Quadruplex : Stem-Loop Equilibrium and Promotes miRNA Processing(A) Schematic representation of a guanine tetrad, highlighting Hoogsteen base pairing involving the N7 of guanosine that stabilizes the G-quadruplex structure, together with a stabilizing monovalent cation (M+, usually potassium). Both 7-methylguanosine and 7-deaza-guanosine are able to destabilize the hydrogen bond involving N7.(B) Illustration depicting the pri-miRNA hairpins used in the following experiments.(C) Thermal denaturation studies of RNA oligonucleotides as described in (B). While GG-to-DAG-DAG mutation at the D1 position does not significantly affect the contribution of G4 in the G4 : stem-loop equilibrium, GG-to-DAG-DAG mutation at the D1 position and a single G11-to-DAG mutation affect the contribution of rG4 in the structural equilibrium by shifting it toward the hairpin form.(D) First derivative plot of the denaturation experiment in (C) helps visualize the decrease in rG4 contribution to the equilibrium (red arrow).(E) qRT-PCR showing the levels of let-7e-5p 72 h after transfection with either WT, D1, D2, or G11 oligonucleotides. The average of six independent transfections ± SDs is shown (∗∗p < 0.01, ∗∗∗p < 0.001, two-tailed t test).(F) Western blot showing the rescue of HMGA2 upregulation upon transfection of D2, but not WT let-7e primary hairpin in A549 METTL1 knockdown cells. Two representative biological replicates of a total of three independent experiments are shown.

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• WB

CiteAb

Western blot - Anti-METTL1 antibody (AB157097)

Western Blotting using Anti-METTL1 antibody, ab157097. Publication image from Balasubramanian, S. et al., 2019, Mol Cell, 31031083. Legend direct from paper.

METTL1 Catalytic Activity Regulates HMGA2 Expression in a let-7-Dependent Manner(A) Schematic of HMGA2 3′ UTR showing the enrichment of evolutionarily conserved target sites of several m7G-containing miRNAs (OR = 5.46, p = 0.001).(B) HMGA2 expression was measured by qRT-PCR in A549 cells infected with METTL1-specific (sh1, sh2) or control (Scr) TET-inducible shRNAs 5 days after doxycycline treatment. The average of six biological replicates ± SDs is shown (∗∗∗p < 0.001, two-tailed t test).(C) Western blot showing METTL1, HMGA2, and β-tubulin protein levels in A549 cells infected with METTL1-specific (sh1, sh2) or control (Scramble) TET-inducible shRNAs 5 days after doxycycline treatment. Two representative biological replicates of a total of four are shown.(D) Western blot showing METTL1 downregulation upon transfection with METTL1-specific siRNAs in A549 cells stably expressing a luciferase cDNA with Hmga2 3′ UTR. Two independent transfections of a total of four replicates are shown.(E) Luciferase fluorescence levels upon METTL1 downregulation in A549 cells stably expressing a luciferase cDNA with Hmga2 3′ UTR as a reporter. Red and gray bars indicate luciferase levels in the presence of either WT Hmga2 3′ UTR or of a variant in which all 7 let-7 seed sequences have been mutated, respectively. The plot shows the average of four independent transfections ± SDs (∗∗∗p < 0.001, two-tailed t test).(F) Western blot showing the rescue of HMGA2 upregulation upon transfection with let-7e-5p mature miRNA in METTL1 knockdown A549 cells. Two independent transfection replicates of a total of four are shown.(G) Western blot showing the rescue of HMGA2 upregulation upon the overexpression of WT, but not catalytically inactive METTL1, in A549 METTL1 knockdown cells. Two representative biological replicates of a total of five independent infections are shown.See also Figure S4.

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