Journal: Nucleic Acids Research

Article Title: HSP90 and Aha1 modulate microRNA maturation through promoting the folding of Dicer1

doi: 10.1093/nar/gkac528

Figure Lengend Snippet: Genetic depletion of Aha1 led to diminished levels of Dicer1, and Aha1 interacts with Dicer1 in HEK293T cells. ( A ) HEK293T cells treated with shAha1 and shControl were transfected with the HSP90–APEX plasmid for 24 h and treated with biotin phenol for 30 min and H 2 O 2 for 1 min, followed by cell lysis and streptavidin affinity pulldown, and the whole-cell lysate and pulldown lysate were used for western blot analysis. ( B ) The change in expression level of Dicer1 in the input panel of (A). ( C ) The alteration in the level of Dicer1 protein in the proximity proteome of HSP90 based on the IP panel of (A). ( D ) The lysates of HEK293T cells with 3× Flag tag on the C-terminus of endogenous HSP90 were used for Flag affinity purification, and the immunoprecipitates were employed to monitor the levels of HSP90-Flag and Dicer1 proteins by western blot. ( E ) HEK293T cells were transfected with the Flag-tagged Aha1 plasmid for 24 h, followed by cell lysis and Flag affinity pulldown, and the whole-cell lysate and pulldown lysate were used for western blot analysis. Dicer1 level in panel (B) was quantified from band intensities using ImageJ and normalized to GAPDH first and then displayed relative to the level in HEK293T shControl cells. Dicer1 level in panel (C) was quantified from band intensities using ImageJ and was displayed relative to the level in HEK293T shControl cells. The data represented the mean ± SD ( n = 3). The P values were calculated using unpaired, two-tailed Student’s t -test: **, 0.001 ≤ P < 0.01; ***, P < 0.001.

Article Snippet: The coding sequences of wild-type Aha1, Aha1-E67K and Aha1-Δ20, where the first 20 amino acids were deleted, were cloned into the BamHI and EcoRI sites of the pRK7 3× Flag vector (Addgene, plasmid #8996). pCAGGS-Flag-hsDicer1 was purchased from Addgene (#41584).

Techniques: Transfection, Plasmid Preparation, Lysis, Western Blot, Expressing, FLAG-tag, Affinity Purification, Two Tailed Test

Journal: Chemical research in toxicology

Article Title: Aha1 Is an Autonomous Chaperone for SULT1A1.

doi: 10.1021/acs.chemrestox.2c00167

Figure Lengend Snippet: Figure 2. SULT1A1 interacts with Aha1. (a) HEK293T cells with genetic depletion of Aha1 (shAha1) and shCtrl cells were transfected with the HSP90-APEX plasmid for 24 h and then incubated with biotin phenol for 30 min and H2O2 for 1 min followed by cell lysis and streptavidin affinity pull-down; the whole-cell lysate and pull- down lysate were employed for Western blot analysis. (b) Change in the expression level of SULT1A1 in the input panel of (a). (c) Alteration in the level of SULT1A1 in the proximity proteome of HSP90 based on the IP panel of (a). (d) The lysates of HEK293T cells with a 3× Flag tag on the C terminus of endogenous HSP90 were employed for immunoprecipitation using an anti-Flag antibody, and the immunoprecipitates were used to monitor the levels of HSP90-Flag and SULT1A1 proteins by Western blotting. (e) HEK293T cells were transfected with the Flag-tagged Aha1 plasmid for 24 h followed by cell lysis and affinity purification using anti-Flag agarose. The whole-cell lysate and pull-down lysate were employed for Western blotting. SULT1A1 protein levels in (b) were quantified from band intensities using ImageJ and normalized to GAPDH first and then displayed relative to the level in HEK293T shCtrl cells. SULT1A1 levels in (c) were quantified from band intensities using ImageJ and were displayed relative to the level in HEK293T shCtrl cells. The data represent the mean ± S.D. (n = 3). The p values were calculated using unpaired, two-tailed Student’s t-test: **, 0.001 ≤p < 0.01; ***, p < 0.001.

Article Snippet: Control shRNA with a hairpin sequence of 5′-CCT AAG GTT AAG TCG CCC TCG CTC TAG CGA GGG CGA CTT AAC CTT AGG-3′ (Addgene, Cambridge, MA, USA) was used as a negative control, as described previously.26 The shRNAs for Aha1 depletion, the HSP90-APEX plasmid, and the pRK7 3× Flag plasmids for expressing wild-type Aha1, Aha1-Δ20 (with the first 20 amino acids of Aha1 being deleted), and Aha1-E67K were described recently.25 The coding sequence of SULT1A1 was cloned into the BamHI and EcoRI sites of the pRK7 3× Flag vector (Addgene, plasmid no. 8996), and the sequence of the resulting plasmid was confirmed by Sanger sequencing.

Techniques: Transfection, Plasmid Preparation, Incubation, Lysis, Western Blot, Expressing, FLAG-tag, Immunoprecipitation, Affinity Purification, Two Tailed Test

Journal: Chemical research in toxicology

Article Title: Aha1 Is an Autonomous Chaperone for SULT1A1.

doi: 10.1021/acs.chemrestox.2c00167

Figure Lengend Snippet: Figure 1. APEX2-based proximity proteomics revealed Aha1-mediated interactions between HSP90 and SULT1A1. (a) Extracted-ion chromatograms (XIC) and (b) corresponding quantification results for a unique peptide of SULT1A1 identified in the proximity proteome of HSP90. Proximity labeling was performed in the control shRNA (shCtrl)-treated or Aha1 knockdown (shAha1) cells. The peak area of the SULT1A1 peptide from Aha1 knockdown cells was normalized to that of the control. Plotted are the means ± S.D. of results obtained from three biological replicates. The p values were calculated using two-tailed, unpaired t-tests (**, 0.01 < p < 0.05; ***, 0.001 < p < 0.01). (c) Representative MS/MS spectrum for a unique peptide of SULT1A1 identified in control shRNA-treated cells.

Article Snippet: Control shRNA with a hairpin sequence of 5′-CCT AAG GTT AAG TCG CCC TCG CTC TAG CGA GGG CGA CTT AAC CTT AGG-3′ (Addgene, Cambridge, MA, USA) was used as a negative control, as described previously.26 The shRNAs for Aha1 depletion, the HSP90-APEX plasmid, and the pRK7 3× Flag plasmids for expressing wild-type Aha1, Aha1-Δ20 (with the first 20 amino acids of Aha1 being deleted), and Aha1-E67K were described recently.25 The coding sequence of SULT1A1 was cloned into the BamHI and EcoRI sites of the pRK7 3× Flag vector (Addgene, plasmid no. 8996), and the sequence of the resulting plasmid was confirmed by Sanger sequencing.

Techniques: Labeling, Control, shRNA, Knockdown, Two Tailed Test, Tandem Mass Spectroscopy