Journal: bioRxiv
Article Title: Separable roles for Microprocessor and its cofactors ERH and SAFB1/2 during microRNA cluster assistance
doi: 10.1101/2025.09.09.675111
Figure Lengend Snippet: (A) Deletion and/or depletion of ERH and SAFB1/2 in HEK293T cells. ERH mutant cells were generated by shRNA-mediated knockdown in an ERH[+/-] heterozygous cell line. SAFB1/2 double mutant cells were generated by knockdown of SAFB1 in a SAFB1[+/-], SAFB2[-/-] cell line. ERH+SAFB1/2 triple mutant cells were generated by knockdown of ERH and SAFB1 in an ERH[+/-]; SAFB1[+/-], SAFB2[-/-] cell line. Western blot validates strong loss of ERH and SAFB1 proteins and absence of SAFB2 in the respective mutant genotypes. β-tubulin was probed as control. (B) Northern blotting to assay processing of mir-144/451 cluster or solo mir-451 in wildtype or cofactor depleted HEK293T cells. Co-transfected mir-375 and endogenous let-7a and U6 snRNAs were probed as controls. RNA size markers (nt) are shown at left. Microprocessing of suboptimal pri-mir-451 , but not optimal pri-mir-144 and pri-mir-375 , is significantly impaired in ERH and/or SAFB1/2 mutant cells. However, lack of the neighboring optimal mir-144 hairpin ablated pri-mir-451 processing, even with all the cofactors present. (C) Rescue of pri-mir-451 processing in mutant cells. While ERH overexpression can restore mir-451 biogenesis in ERH mutant cells, only SAFB2 overexpression restored mir-451 biogenesis in SAFB1/2 and ERH+SAFB1/2 mutant cells.
Article Snippet: The blots were probed for 2 hrs at room temperature or overnight at 4°C with Rabbit polyclonal anti-SAFB1 antibody (Cat #A300-812A) diluted to 1:2000, or Rabbit polyclonal anti-SAFB2 antibody (Cat #A301-112A) diluted to 1:2000, or Rabbit polyclonal anti-ERH antibody (Cat #PA5-21388) diluted to 1:2000, or mouse monoclonal anti-β-tubulin antibodies (DSHB) diluted to 1:2000, and then incubated with a secondary antibody conjugated to horseradish peroxidase diluted to 1:5000.
Techniques: Mutagenesis, Generated, shRNA, Knockdown, Western Blot, Control, Northern Blot, Transfection, Over Expression
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![(A) Deletion and/or depletion of ERH and SAFB1/2 in HEK293T cells. ERH mutant cells were generated by shRNA-mediated knockdown in an ERH[+/-] heterozygous cell line. SAFB1/2 double mutant cells were generated by knockdown of SAFB1 in a SAFB1[+/-], SAFB2[-/-] cell line. ERH+SAFB1/2 triple mutant cells were generated by knockdown of ERH and SAFB1 in an ERH[+/-]; SAFB1[+/-], SAFB2[-/-] cell line. Western blot validates strong loss of ERH and SAFB1 proteins and absence of SAFB2 in the respective mutant <t>genotypes.</t> <t>β-tubulin</t> was probed as control. (B) Northern blotting to assay processing of mir-144/451 cluster or solo mir-451 in wildtype or cofactor depleted HEK293T cells. Co-transfected mir-375 and endogenous let-7a and U6 snRNAs were probed as controls. RNA size markers (nt) are shown at left. Microprocessing of suboptimal pri-mir-451 , but not optimal pri-mir-144 and pri-mir-375 , is significantly impaired in ERH and/or SAFB1/2 mutant cells. However, lack of the neighboring optimal mir-144 hairpin ablated pri-mir-451 processing, even with all the cofactors present. (C) Rescue of pri-mir-451 processing in mutant cells. While ERH overexpression can restore mir-451 biogenesis in ERH mutant cells, only SAFB2 overexpression restored mir-451 biogenesis in SAFB1/2 and ERH+SAFB1/2 mutant cells.](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_11/10__1101_slash_2025__09__09__675111/10__1101_slash_2025__09__09__675111___F1.large.jpg)