Journal: bioRxiv

Article Title: HAP40 functions as a proteostasis regulator by controlling huntingtin interactions and its release into the extracellular space

doi: 10.64898/2026.03.31.715351

Figure Lengend Snippet: A) Schematic representation of the BRET assay workflow. Expression vectors encoding c-myc-NL and PA-mCit tagged proteins were co-transfected into HEK293 cells. Binary interactions were detected in vivo using in-cell bioluminescence resonance energy transfer (BRET). B) BRET ratios of binary interactions between NanoLuc- and mCitrine-tagged full-length HTT Q23 , HAP40, and respective controls. Data are presented as mean ± SD (n=2), each with three technical replicates. Statistical significance was determined via two-way ANOVA followed by Bonferroni’s multiple-comparisons test. C) Left: Cryo-EM structure of HTT-HAP40 (6X9O). HAP40 is shown in red and HTT in blue. Right: Zoomed-in view displaying the five conserved HAP40 amino acid residues (gold: E316, E317, E331, D333, E335) and the three HAP40 mutant variants generated based on the number of amino acids converted into lysine. D) BRET ratios of binary interactions between c-myc-NL-HTT Q23 and PA-mCit-HAP40 (wild-type and mutants). Data are presented as mean ± SD, n=2, each with three technical replicates. Statistical significance was determined via two-way ANOVA followed by Dunnett’s multiple-comparisons test. E) Top: Linear schematic representation of resolved (blue) and unresolved (red) segments of the Cryo-EM structure of HTT-HAP40 (6X9O), indicating the mCitrine (mCit) insertion site. Below: schematic representation of the HTT intramolecular BRET sensor (NL-HTT Q23 (2686-mCit) ), displaying the tagging positions of NanoLuc at the N-terminus and mCitrine between amino acids 2686 and 2687 of human HTT. F) Cryo-EM structures of apo-HTT (6RMH) and HTT-HAP40 (6EZ8), illustrating the distances between resolved residues near the mCitrine insertion site and the N-terminus of HTT. Distances are shown in angstroms (Å). The relative positions of NanoLuc and mCitrine are indicated, along with predicted differences in BRET signal based on donor-acceptor distance. G) BRET saturation assay with co-transfection of the HTT intramolecular BRET sensor (150 ng, pcDNA-NL-HTT Q23 (2686-mCit) ) and increasing amounts of pcDNA-c-myc-HAP40 (wild-type; 0-10 ng) in HEK293-HAP40KO cells. The plot displays BRET ratio values (dashed line) and luminescence values (bar graph). Data are presented as mean ± SD (n=2). Statistical significance was determined via one-way ANOVA followed by Dunnett’s multiple-comparisons test. Corresponding immunoblots related to BRET assay using anti-c-myc, anti-GFP, and anti-TIM23 (loading control) antibodies are shown below. H) BRET saturation assay using the HTT intramolecular BRET sensor with increasing amounts of c-myc-HAP40 (5M mutant). Experimental conditions and statistical analysis were performed as described in G .

Article Snippet: The following primary antibodies were applied overnight at 4°C: rabbit anti-HAP40 (Atlas Antibodies, HPA046960, 1:1000), anti-HTT D7F7 antibody (Cell Signaling, 5656, 1:1000), anti-HTT MAB2166 antibody (Millipore, MAB2166, 1:500), anti-HTT MW1 antibody (DSHB, MW1, 1:500), mouse anti-SQSTM1(p62) (abcam, ab56416, 1:1000), rabbit anti-LC3B (abcam, ab192890, 1:1000), rabbit anti-UBB (PTG, 10201-2-AP, 1:1000), rabbit anti-Cathepsin D (Cell Signaling 69854, 1:1000), rabbit anti-STX17 (PTG 17815-1-AP, 1:1000), mouse anti-FLAG (Sigma, F3165, 1:1000), mouse anti-mNeonGreen (Chromotek, 32F6, 1:200), rabbit anti-GFP (Abcam, ab290, 1:2500), mouse anti-c-Myc (Merck, M4439, 1:2000), mouse anti-Actin (abcam, ab8224, 1:1000), mouse anti-Tubulin (Sigma, T6074, 1:80,000), mouse anti-TIM23 (BD Biosciences, 611223, 1:1000), and mouse anti-GAPDH (Santa Cruz, sc-47724, 1:1000).

Techniques: Bioluminescence Resonance Energy Transfer, Expressing, Transfection, In Vivo, Cryo-EM Sample Prep, Mutagenesis, Generated, Saturation Assay, Cotransfection, Western Blot, Control

Journal: bioRxiv

Article Title: c-MYC is an aggregation-prone, amyloidogenic protein

doi: 10.64898/2026.03.12.711438

Figure Lengend Snippet: (A) Detection of detergent-soluble and - insoluble c-MYC proteins by immunoblotting in HeLa cells pre-treated with 30 μM CR, followed by HS at 43°C for 30 min (representative images of three independent experiments). (B) and (C) Immunoprecipitation of either exogenously expressed HA-c-MYC or endogenous c-MYC proteins in HeLa cells by A11 antibodies (representative images of three independent experiments). (D) Detection of endogenous c-MYC proteins in HeLa cells by PLA using both the goat anti-c-MYC Ab and the rabbit anti-AO (A11) Ab. Nuclei are counterstained with Hoechst 33342. Scale bar: 10 μm. (E)-(G) Detection of endogenous c-MYC proteins in human prostate adenocarcinoma, hepatocellular carcinoma, and Alzheimer’s disease brain tissues by brightfield PLA using both the goat anti-c-MYC Ab and the rabbit anti-AO (A11) Ab. Scale bar: 100 μm for low magnification and 10 μm for high magnification. (H): Quantitation of endogenous c-MYC recognized by both the goat anti-c-MYC Ab and the rabbit anti-AO (A11) Ab through In-Cell PLA ELISA in HeLa cells treated with CR (mean ± SD, n=3 independent experiments, One-way ANOVA).

Article Snippet: All antibodies were purchased commercially, including rabbit polyclonal anti-amyloid oligomer (A11) Ab (cat#SPC-506, StressMarq Biosciences Inc.), rabbit monoclonal anti-c-MYC/N-MYC (D3N8F) Ab (cat#13987, Cell Signaling Technology), rabbit monoclonal ani-MAX (E6F6Y) Ab (cat#17471, Cell Signaling Technology), mouse monoclonal anti-HSC/HSP70 (W27) Ab (cat#sc-24, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-HSP90α/β (F-8) Ab (cat#sc-13119, Santa Cruz Biotechnology Inc.), rabbit monoclonal anti-cleaved caspase 3 (Asp175) (D3E9) Ab (cat#9579, Cell Signaling Technology), goat polyclonal anti-c-MYC Ab (cat#AF3696, R&D Systems Inc.), mouse monoclonal anti-HA Ab (cat#901513, BioLegend Inc.), mouse monoclonal anti-βActin (GT5512) Ab (cat#GTX629630, GeneTex Inc.), normal rabbit IgG (cat#02-6102,ThermoFisher Scientific Inc.), Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (cat#111-035-144, Jackson ImmunoResearch Inc.), Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) (cat#115-035-003, Jackson ImmunoResearch Inc.), and Peroxidase IgG Fraction Monoclonal Mouse Anti-Goat IgG, light chain specific (cat#205-032-176, Jackson ImmunoResearch Inc.).

Techniques: Western Blot, Immunoprecipitation, Quantitation Assay, Enzyme-linked Immunosorbent Assay