mouse monoclonal anti α tubulin  (Proteintech)

Journal: bioRxiv

Article Title: Capturing Translation in Action with Protein Synthesis Profiling

doi: 10.1101/2025.11.17.688896

Figure Lengend Snippet: a, Structural model of APEX2-eEF2 bound to the ribosome. The large ribosomal subunit is shown in cyan, the small subunit in green, APEX2-eEF2 in magenta, and mRNA as a black line with the red circles indicating the alkyne modification. The blue sphere marks the ∼25 nm labeling radius of APEX2. b, Expression constructs for APEX2 and APEX2-eEF2 under the TDH3 promoter. c, Western blot detection of APEX2 and APEX2-eEF2 expression. Lanes: control (empty vector), APEX2 (27 kDa), and APEX2-eEF2 (118 kDa). Tubulin (Tub) was probed as a loading control. d, Schematic of the RNA tagging workflow. Yeast cells were incubated with alkyne-phenol (30 min), followed by H₂O₂ (5 min). After quenching, total RNA was extracted, conjugated to biotin-azide via click chemistry, and enriched with streptavidin beads. Both total and enriched RNA were used for preparing Illumina sequencing libraries. e, Agarose gel analysis of total RNA from control, APEX2, and APEX2-eEF2 cells. The presence of intact 25S and 18S rRNA bands indicates high RNA quality. M, molecular weight ladder. f, Detection of alkyne-labeled RNAs by conjugation with fluorescein-azide. Total RNA from control, APEX2, and APEX2-eEF2 cells was subjected to click chemistry and analyzed by agarose gel electrophoresis. Fluorescence was detected using a Typhoon imager. g, The same gel as in F, stained with SafeStain to verify equal RNA loading. h, Quantification of fluorescein-labeled RNA signal. The bar graph shows fluorescence intensity normalized to total RNA, averaged across two independent experiments. i, Gel-shift assay of biotin-labeled RNAs incubated with anti-biotin-AF488 antibody. RNAs from control, APEX2, and APEX2-eEF2 cells were conjugated with biotin-azide, bound by antibody, and resolved on an agarose gel. Antibody-RNA complexes are indicated by the black bar. Lanes: M, molecular weight ladder; control, RNA from control cells; APEX2, RNA from APEX2-expressing cells; APEX2-eEF2, RNA from APEX2-eEF2-expressing cells; Ab, antibody only.

Article Snippet: The blot was reprobed with mouse anti-alpha-tubulin monoclonal antibody (1:8000 dilution) (DSHB Cat# 12G10) in 5% milk for 2 hours at room temperature, followed by the HRP-conjugated anti-mouse IgG secondary antibody (1:10,000 dilution) and detected as described above.

Techniques: Modification, Labeling, Expressing, Construct, Western Blot, Control, Plasmid Preparation, Incubation, Illumina Sequencing, Agarose Gel Electrophoresis, Molecular Weight, Conjugation Assay, Fluorescence, Staining, Gel Shift

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