anti stomatin polyclonal antibody  (Proteintech)

Journal: bioRxiv

Article Title: Structural Role of Stomatin in Organizing Functional Membrane Microdomains

doi: 10.1101/2025.08.30.673307

Figure Lengend Snippet: (A–D) Cryo-EM density map of the STOM-single oligomer purified in DDM. The complex assembles into a bowl-shaped hexadecamer consisting of 16 subunits. The SPFH1 domain and intramembrane hairpin span ∼35 Å in height, the SPFH2 domain ∼45 Å, and the coiled-coil (CC) region ∼35 Å (A–B). The overall diameter of the hexadecamer is ∼130 Å (C), while the membrane area encircled by the SPFH1 domains measures ∼123 Å (D). (E–H) Cryo-EM density map of the STOM-double oligomer purified in DDM. The polymer consists of two bowl-shaped assemblies joined base-to-base via their N-terminal domains (NTDs). The height of each hexadecameric layer is consistent with that of STOM-single (E–F). The overall diameter of STOM-double is ∼110 Å (G), and the enclosed membrane area defined by the SPFH1 domains is ∼118 Å (H). (I–J) Atomic model of stomatin built on the cryo-EM density maps of STOM-single (I) and STOM-double (J). (K) Structural comparison of atomic models derived from the STOM-single (green) and STOM-double (orange) maps. The C-terminal regions align well, whereas a significant displacement is observed at the N-terminus, with a maximal shift of ∼12 Å in the hairpin domain. (L) Schematic representation of stomatin domain organization (top) and corresponding atomic model with topological diagram (bottom). NTD, N-terminal domain. HP, hairpin structure. NTL, N-terminal loop. SPFH1, SPFH1 domain. SPFH2, SPFH2 domain. CC1, coiled-coil domain 1. CC2, coiled-coil domain 2. CTD, C-terminal domain.

Article Snippet: Primary antibody incubation was performed overnight at 4°C using anti-stomatin polyclonal antibody (Proteintech) diluted in blocking solution.

Techniques: Cryo-EM Sample Prep, Purification, Membrane, Polymer, Comparison, Derivative Assay

Journal: bioRxiv

Article Title: Structural Role of Stomatin in Organizing Functional Membrane Microdomains

doi: 10.1101/2025.08.30.673307

Figure Lengend Snippet: (A–D) Cryo-EM density maps of the bowl-bottom structure. From bottom to top: the outermost CC1 domain forms a layer with a diameter of ∼55 Å (D); the second layer consists of CC2, with a diameter of ∼43 Å (C); the innermost layer is formed by the β8-barrel of the CTD, with a diameter of ∼25 Å (B); and the lid structure beyond the β8-barrel contains a central hole with a diameter of ∼17 Å (A). (E) Hydrophilic and hydrophobic surface representation of the bowl-bottom region, revealing a central hydrophobic channel with a minimum diameter of ∼19 Å. (F–H) Cryo-EM density map (F) and atomic model (G–H) showing that adjacent β8-barrels alternate between inward- and outward-facing conformations. (I–J) Atomic model illustrating distinct secondary structures of the β8 segments: one subunit adopts an inward-facing α-helix, while the neighboring subunit forms an outward-projecting loop. (K) Structural model demonstrating that E227 in CC1 forms stabilizing salt bridges with acidic residues in CC2 and CC1 of adjacent subunits, thereby supporting oligomerization. (L–M) Mutational analyses of oligomerization. Protein expression was verified in cells (L), and 10%–50% glycerol gradient fractionation was used to examine the distribution of stomatin proteins (M). Stomatin-WT distributes into both high-molecular-weight multimers and low-molecular-weight monomers/oligomers. Two conserved proline residues (P200 and P245) are required for oligomerization, as their mutations (P200A, P245A) abolished polymer formation. Truncation of the CTD also impaired multimerization, with a strong effect observed at residues 274–276 (highlighted in I). D274N increased the proportion of multimers, consistent with reduced inter-subunit charge repulsion.

Article Snippet: Primary antibody incubation was performed overnight at 4°C using anti-stomatin polyclonal antibody (Proteintech) diluted in blocking solution.

Techniques: Cryo-EM Sample Prep, Expressing, Fractionation, High Molecular Weight, Molecular Weight, Polymer

Journal: bioRxiv

Article Title: Structural Role of Stomatin in Organizing Functional Membrane Microdomains

doi: 10.1101/2025.08.30.673307

Figure Lengend Snippet: (A) Cryo-EM micrograph of stomatin reconstituted into POPC:DOPS:cholesterol (8:1:1) liposomes, showing the formation of stomatin clusters on the membrane. Scale bar, 50 nm. (B) STOM particles located in low-curvature membrane regions were selected for 2D classification (box size, 640 pixel). STOM particles appeared regularly arranged on the membrane. (C–D) U-2 OS cells were incubated with 25-NBD-cholesterol–BSA to replace cholesterol and subsequently immunostained for endogenous stomatin. Confocal microscopy revealed cholesterol enrichment at stomatin-positive sites (first three pictures of C; scale bar, 10 μm). STED super-resolution imaging confirmed cluster-like stomatin distribution (Zoom picture of C; scale bar, 2 μm). Cluster size and number were quantified in FIJI (D), showing diameters primarily within 50–150 nm, with some clusters >300 nm. Data represent three independent experiments (n = 27). (E–F) Polarization-resolved SIM analysis of U-2 OS cells expressing GFP-STOM. Representative images show GFP localization and Nile Red polarization mapping (E; scale bar, 2 μm). Blue arrows indicate analyzed membrane regions. Pseudo-color coding of Nile Red indicates lipid order: red, higher modulation depth (greater order); yellow, lower modulation depth (reduced order). Quantification of modulation depth (F) demonstrated significantly increased order in GFP-STOM-WT compared with GFP-PM (P < 0.0001) and GFP-STOM-P245A (P < 0.0001), whereas GFP-PM and GFP-STOM-P245A showed no significant difference (P = 0.6955). Data were obtained from three independent experiments; each point represents one membrane region. Statistical analysis was performed using an unpaired t-test (ns, not significant; ****, P < 0.0001). (G) Transient dehydration of A549 cells stably expressing GFP-STOM-WT was used to mimic stomatin-induced membrane remodeling. Removal of DMEM for 5 min followed by re-addition caused cell shrinkage, leaving GFP-STOM–enriched vesicles at the cell periphery. Adjacent vesicles rapidly fused with stomatin accumulation at the fusion sites (yellow arrows), accompanied by membrane fragmentation and reorganization at cluster edges.

Article Snippet: Primary antibody incubation was performed overnight at 4°C using anti-stomatin polyclonal antibody (Proteintech) diluted in blocking solution.

Techniques: Cryo-EM Sample Prep, Liposomes, Membrane, Incubation, Confocal Microscopy, Imaging, Expressing, Stable Transfection