Journal: The FASEB Journal

Article Title: Chromogranin A preferential interaction with Golgi phosphatidic acid induces membrane deformation and contributes to secretory granule biogenesis

doi: 10.1096/fj.202000074r

Figure Lengend Snippet: FIGURE 1 CgA interacts with PA through a putative PABD and Golgi PA is involved in CgA secretory granule biogenesis. A, Protein-lipid overlay assay in the presence of recombinant CgA (500 ng/mL) using commercial membrane strips where 100 pmol/spot of the following lipids are adsorbed: triglycerides, diacylglycerol (DAG), phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylglycerol (PG), cardiolipin, phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PI(4) P), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3), cholesterol, sphingomyelin, or 3-sulfogalactosylceramide (sulfatide). After the overlay, the membrane was immunostained for CgA using the anti-WE-14 antibody, and revealed using a chemiluminescence kit. Plotted are means of CgA binding intensity expressed as percentage normalized to control (blank) ± SEM (n = 3). ***P < .001, (ANOVA) one-way analysis of variance test with Bonferroni's comparison test. B, Involvement of Golgi PA in the formation of CgA-containing secretory granules in PC12 cells. Cells expressing wild-type PDE4A1 coupled with GFP (PDE4A1(PABD)) or PABD-mutated PDE4A1 coupled with GFP (PDE4A1(PABD Mut)) are surrounded by a dashed line. Cells were immunolabeled with anti-CgA antibody and their fluorescence was examined using confocal microscopy. Representative confocal microscopy sections throughout the cells are shown. Values for the intensity of CgA staining and the number of granules per cell are plotted as the means ± SD (n = 2; 40 cells per condition). **P < .01, Mann- Whitney test. The scale bar represents 20 µm. Western blots show expression of CgA and α-tubulin as loading control. C, Human CgA (hCgA) sequence showing a region of 18 amino acids (364-381), encompassing a positive charge cluster and hydrophobic residues that could adopt an α-helical conformation, delimited by arrows, suggesting its function as PA-binding domain (PABD). D, PA-binding profile of the putative human CgA PABD. Amphipathic α-helix projection of the core 18 amino acids of the PABD of CgA obtained with Heliquest software. Arrow indicates hydrophobic moment. Red N and C indicate the beginning and the end of PABD amino acid sequence. E, Model of the putative human CgA PABD. Ribbon corresponds to the α-helix. The structure was determined using MacPyMOL (v1.74). F, Characterization of the PA-binding capacity of human CgA through the putative PABD. Semi-quantitative fluorescent liposome assays with fluorescent liposomes (5% PE-NBD, 95% DOPC) or PA-containing fluorescent liposomes (5% PE-NBD, 85% DOPC, 10% PA mix) and GST-CgA-PABD constructs linked to GSH-sepharose beads. The binding of liposomes with CgA-PABD constructs was monitored by fluorimetry. Results are presented as means ± SD (triplicate measurements; n = 3)

Article Snippet: Rat pheochromocytoma PC12 cells (American Type Culture Collection; CRL 1721) were routinely grown in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, Thermo Fisher Scientific) supplemented with 5% of FBS, 10% of sterile-filtered HyClone Donor Equine serum (GE Healthcare, Life Sciences), 100 U mL−1 penicillin, 100 μg mL−1 streptomycin (Gibco, Thermo Fisher Scientific) and 1% of L-glutamine 100X (Gibco, Thermo Fisher Scientific), at 37°C in 5% of CO2.

Techniques: Protein-lipid Overlay Assay (PLOA), Recombinant, Membrane, Binding Assay, Control, Comparison, Expressing, Immunolabeling, Fluorescence, Confocal Microscopy, Staining, MANN-WHITNEY, Western Blot, Sequencing, Software, Liposomes, Construct

Journal: The FASEB Journal

Article Title: Chromogranin A preferential interaction with Golgi phosphatidic acid induces membrane deformation and contributes to secretory granule biogenesis

doi: 10.1096/fj.202000074r

Figure Lengend Snippet: FIGURE 2 Same predominant PA species are found in Golgi and secretory granule membranes from COS7-WT, COS7-CgA, and PC12 cells. A, The level of distinct PA species was measured in fractions of secretory granule (SG) and Golgi membranes from COS7-CgA cells by duplicate UPLC/MS/MS analysis of two samples (each containing 200 µg of protein) from each fraction. The acyl chain composition of each species is shown on the x axis. The y axis shows the abundance of each species as a percentage of total PA in the sample. B, PA levels were measured in fractions of Golgi membranes from COS7-WT or COS7-CgA cells by duplicate UPLC/MS/MS analysis of two samples (each containing 200 µg of protein) from each fraction. The acyl chain composition of each species is shown on the x axis. The y axis shows the abundance of each species as a percentage of total PA in the sample. C, PA levels were measured in secretory granule-containing fractions from PC12 or COS7-CgA cells by duplicate UPLC/MS/MS analysis of two samples (each containing 200 µg of protein) from each fraction. The acyl chain composition of each species is shown on the x axis. The y axis shows the abundance of each species as a percentage of total PA in the sample

Article Snippet: Rat pheochromocytoma PC12 cells (American Type Culture Collection; CRL 1721) were routinely grown in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, Thermo Fisher Scientific) supplemented with 5% of FBS, 10% of sterile-filtered HyClone Donor Equine serum (GE Healthcare, Life Sciences), 100 U mL−1 penicillin, 100 μg mL−1 streptomycin (Gibco, Thermo Fisher Scientific) and 1% of L-glutamine 100X (Gibco, Thermo Fisher Scientific), at 37°C in 5% of CO2.

Techniques: Tandem Mass Spectroscopy

Journal: The FASEB Journal

Article Title: Chromogranin A preferential interaction with Golgi phosphatidic acid induces membrane deformation and contributes to secretory granule biogenesis

doi: 10.1096/fj.202000074r

Figure Lengend Snippet: FIGURE 8 Inhibition of PLD-mediated PA synthesis alters secretory granule biogenesis. A, COS7-CgA cells were treated or not with 75 nM FIPI for 6 h, fixed and immunolabeled with anti-GM130 and anti-CgA antibodies. Representative confocal microscopy sections throughout the cells are shown. The scale bar represents 20 µm. Values for the number of CgA secretory granules are plotted as the means ± SEM. (n = 3; 60 cells per condition). ***P < .001, Mann-Whitney test. Western blots show expression of CgA and α-tubulin as loading control. B, PC12 cells were treated or not with 75 nM FIPI for 6 h, fixed and immunolabeled with anti-GM130 and anti-CgA antibodies. Representative confocal microscopy sections throughout the cells are shown. The scale bar represents 10 µm. Values for the number of CgA secretory granules are plotted as the means ± SEM. (n = 3; 60 cells per condition). Mann-Whitney test. Western blots show expression of CgA, CgB, and SgII proteins, and α-tubulin as loading control. C, Electron microscopy analysis of chromaffin cells from WT (control) and Pld1−/− mice. White arrows indicate representative secretory granules. The scale bars represent 2 µm. D, Quantification of the number of secretory granules (SG) shown in panel (A) (n = 6 mice per condition; 50 cells per mouse adrenal medulla). *P < .05, Mann-Whitney test. Means ± SEM are plotted. E, Quantification of the dense core diameter of secretory granules (SG) shown in panel (A) (n = 6 mice per condition; 950 granules). *P < .05, Mann-Whitney test. Means ± SEM are plotted

Article Snippet: Rat pheochromocytoma PC12 cells (American Type Culture Collection; CRL 1721) were routinely grown in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, Thermo Fisher Scientific) supplemented with 5% of FBS, 10% of sterile-filtered HyClone Donor Equine serum (GE Healthcare, Life Sciences), 100 U mL−1 penicillin, 100 μg mL−1 streptomycin (Gibco, Thermo Fisher Scientific) and 1% of L-glutamine 100X (Gibco, Thermo Fisher Scientific), at 37°C in 5% of CO2.

Techniques: Inhibition, Immunolabeling, Confocal Microscopy, MANN-WHITNEY, Western Blot, Expressing, Control, Electron Microscopy

Journal: Mediators of Inflammation

Article Title: Implication of Ceramide Kinase in Adipogenesis

doi: 10.1155/2017/9374563

Figure Lengend Snippet: CerK is upregulated during adipogenesis. Cells were seeded in 6-well plates (1.2 × 10 5 cells/well), and they were differentiated as described in Materials and Methods. (a) CerK was detected by Western blotting using a specific antibody. Equal loading of protein was assessed with an antibody against GAPDH. Similar results were obtained in each of 4 replicate experiments. (b) Results of the scanning densitometry of exposed film. Data are expressed as arbitrary units of intensity relative to GAPDH and are the mean ± SEM of 4 independent experiments ( ∗ p < 0.05). (c) CerK activity was determined as described in Materials and Methods. Data are expressed as the mean ± SEM of 5 independent experiments ( ∗∗ p < 0.01). (d) C1P levels were determined as described in Materials and Methods. Data are expressed as the mean ± SEM of four independent determinations ( ∗ P < 0.05).

Article Snippet: The GAPDH antibody, nontargeting (negative) siRNA, and ceramide kinase (CerK) siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Activity Assay

Journal: Mediators of Inflammation

Article Title: Implication of Ceramide Kinase in Adipogenesis

doi: 10.1155/2017/9374563

Figure Lengend Snippet: CerK downregulation leads to decreased lipid droplet formation and depletion of TG content in 3T3-L1 cells. The preadipocytes were seeded in 100 mm diameter dishes at 5 × 10 5 cells/plate and incubated for 48 h as indicated in . They were then electroporated in the absence of siRNA (ctrl) or in the presence of negative (scrambled) siRNA (Neg siRNA) or Cerk siRNA, as indicated in Materials and Methods, and were then differentiated up to day 4. (a) CerK knockdown using specific siRNA was confirmed by immunoblotting. Equal loading of protein was assessed with an antibody against GAPDH. Similar results were obtained in each of 3 replicate experiments. (b) Results of the scanning densitometry of exposed film. Data are expressed as arbitrary units of intensity relative to GAPDH and are the mean ± SEM of 3 independent experiments ( ∗ p < 0.05). (c) Light micrographs of adipocytes stained with Oil Red O at day 4 after differentiation are shown. Pictures were taken with a motorized Nikon Eclipse TS100 microscope at 20x magnification. (d) Cells were stained with Oil Red O, and lipid droplets were quantified at day 4 after differentiation as indicated in Materials and Methods. Data are expressed as the mean ± SEM of 5 independent experiments performed in triplicate ( ∗ p < 0.05). (e) Triacylglycerol (TG) content of cells was measured at day 4 after differentiation as described in Materials and Methods. Results are the mean ± SEM of 3 independent experiments performed in triplicate ( ∗ p < 0.05).

Article Snippet: The GAPDH antibody, nontargeting (negative) siRNA, and ceramide kinase (CerK) siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Incubation, Knockdown, Western Blot, Staining, Microscopy

Journal: Mediators of Inflammation

Article Title: Implication of Ceramide Kinase in Adipogenesis

doi: 10.1155/2017/9374563

Figure Lengend Snippet: CERK downregulation leads to inhibition of leptin secretion and inhibition of PPAR- γ expression. The preadipocytes were seeded in 100 mm diameter dishes at 5 × 10 5 cells/plate and incubated for 48 h as indicated in . The cells were then electroporated in the absence of siRNA (ctrl) or in the presence of negative (scrambled) siRNA (Neg siRNA) or Cerk siRNA, as indicated in Materials and Methods. (a) Leptin concentration in supernatants was measured at day 4 after differentiation using an ELISA kit, as described in Materials and Methods. Results are the mean ± SEM of 4 independent experiments performed in duplicate ( ∗ p < 0.05). (b) PPAR γ expression was detected by Western blotting using a specific antibody. Equal loading of protein was assessed with an antibody against GAPDH. Similar results were obtained in each of 5 replicate experiments. (c) Results of scanning densitometry of the exposed film. Data are expressed as arbitrary units of intensity relative to GAPDH and are the mean ± SEM of 5 independent experiments ( ∗ p < 0.05).

Article Snippet: The GAPDH antibody, nontargeting (negative) siRNA, and ceramide kinase (CerK) siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Inhibition, Expressing, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: bioRxiv

Article Title: Structures of human organellar SPFH protein complexes

doi: 10.1101/2025.04.21.649849

Figure Lengend Snippet: a, Domain scheme of Erlin1 and Erlin2. b, Sequence alignment of human Erlin1 and Erlin2, with structural features noted above, colored according to domain. c, Coomassie stain (top) and immunoblotting (bottom) of the Erlin1/2 complex tandemly purified via N-terminally Strep-tagged Erlin1 and C-terminally Flag-tagged Erlin2. PD, pulldown; FT, flow-through; Elu, elution. d, Size exclusion chromatography (SEC) trace, showing absorbance at 280 nm values, of the tandem-purified Erlin1/2 complex as in c. The fractions corresponding to Erlin1/2 as indicated were pooled for cryo-EM analysis.

Article Snippet: HRP-conjugated anti-FLAG M2 (Sigma A8592, 1:5,000), HRP-conjugated StrepTactin (Bio-rad 1610381, 1:5,000), anti-Erlin1 (Proteintech 14898-1-AP, 1:1000 or Abcam ab178687, 1:10,000), anti-Erlin2 (Proteintech 16018-1-AP, 1:4,000), anti-PHB1 (Bethyl A301-873A, 1:5,000), anti-PHB2 (Bethyl A301-873A, 1:5,000), anti-AFG3L2 (Abcam ab68023, 1:500), anti-SPG7 (Novus NBP2-01860, 1:500), and anti-MIRO2 (Abcam ab224089, 1:1,000) were used for immunoblotting.

Techniques: Sequencing, Staining, Western Blot, Purification, Size-exclusion Chromatography, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structures of human organellar SPFH protein complexes

doi: 10.1101/2025.04.21.649849

Figure Lengend Snippet: a, Model and secondary structural element organization of Erlin1 (left) and Erlin2 (right), colored by domain. b, Map-to-model fits for the indicated regions of Erlin1 (top) or Erlin2 (bottom).

Article Snippet: HRP-conjugated anti-FLAG M2 (Sigma A8592, 1:5,000), HRP-conjugated StrepTactin (Bio-rad 1610381, 1:5,000), anti-Erlin1 (Proteintech 14898-1-AP, 1:1000 or Abcam ab178687, 1:10,000), anti-Erlin2 (Proteintech 16018-1-AP, 1:4,000), anti-PHB1 (Bethyl A301-873A, 1:5,000), anti-PHB2 (Bethyl A301-873A, 1:5,000), anti-AFG3L2 (Abcam ab68023, 1:500), anti-SPG7 (Novus NBP2-01860, 1:500), and anti-MIRO2 (Abcam ab224089, 1:1,000) were used for immunoblotting.

Techniques:

Journal: bioRxiv

Article Title: Structures of human organellar SPFH protein complexes

doi: 10.1101/2025.04.21.649849

Figure Lengend Snippet: a, Surface representation of the Erlin1/2 complex model, with two individual subunits colored in pink and blue, and the other subunits colored according to the indicated domain. b, Surface representations of the Erlin1/2 complex model colored by molecular lipophilicity potential (mlp: teal – most hydrophilic, dark goldenrod – most lipophilic, left) or by Coulombic electrostatic potential (right). Orange and red arrowheads note hydrophobic and acidic surfaces. c, Model of the N-terminal transmembrane (TM) and SPFH1 domains of Erlin1 (pink) and Erlin2 (blue) with hydrophobic residues at the endoplasmic reticulum (ER) membrane interface indicated. Aromatic residues are indicated in bold. d, Model of alternating Erlin1 and Erlin2 subunits with the SPFH2 domains colored as in c. Residues involved in intersubunit hydrogen bonds are colored yellow and indicated. Residues that contribute to an acidic patch and N-linked glycosylation sites (N-glyc) are also indicated. e , Model of alternating Erlin and Erlin2 CC1 helices with hydrogen bonding interactions indicated. f, Model as in d with the positions of disease-linked mutations indicated in purple.

Article Snippet: HRP-conjugated anti-FLAG M2 (Sigma A8592, 1:5,000), HRP-conjugated StrepTactin (Bio-rad 1610381, 1:5,000), anti-Erlin1 (Proteintech 14898-1-AP, 1:1000 or Abcam ab178687, 1:10,000), anti-Erlin2 (Proteintech 16018-1-AP, 1:4,000), anti-PHB1 (Bethyl A301-873A, 1:5,000), anti-PHB2 (Bethyl A301-873A, 1:5,000), anti-AFG3L2 (Abcam ab68023, 1:500), anti-SPG7 (Novus NBP2-01860, 1:500), and anti-MIRO2 (Abcam ab224089, 1:1,000) were used for immunoblotting.

Techniques: Membrane

Journal: bioRxiv

Article Title: Structures of human organellar SPFH protein complexes

doi: 10.1101/2025.04.21.649849

Figure Lengend Snippet: a, Clipped side view of the Erlin1/2 complex cryo-EM map with the elements that form four structural layers at the narrow end of the ‘cage’ colored as indicated. b, Top view of the Erlin1/2 complex model with the narrow end colored as in a. c, Isolated elements contributing to the four layers colored as in a. d, Hydrophobic packing and hydrogen bonding interactions involving the CC2 helices of Erlin that facilitate the transitions from the CC1 helices and into β9 of the CT. The CC2 helix of the ‘leftmost’ subunit, when viewed from the outside of the cage, is orange and modeled as Erlin2. The other two subunits are modeled as Erlin1 (pink) and Erlin2 (blue). e, Interactions involving the IPNMF motif in the Erlin CTs and that facilitate the turning of alternate Erlin CTs towards the outer edge of the cage. The leftmost subunit is modeled as Erlin2 and colored according to designations as in a. The other two subunits are modeled as Erlin1 (pink) and Erlin2 (blue). f, Positions of disease-linked mutations (purple) in the CC2 and CT domains of Erlin1 (pink) or Erlin2 (colored as in e or blue), viewed from the outside of the Erlin1/2 complex cage.

Article Snippet: HRP-conjugated anti-FLAG M2 (Sigma A8592, 1:5,000), HRP-conjugated StrepTactin (Bio-rad 1610381, 1:5,000), anti-Erlin1 (Proteintech 14898-1-AP, 1:1000 or Abcam ab178687, 1:10,000), anti-Erlin2 (Proteintech 16018-1-AP, 1:4,000), anti-PHB1 (Bethyl A301-873A, 1:5,000), anti-PHB2 (Bethyl A301-873A, 1:5,000), anti-AFG3L2 (Abcam ab68023, 1:500), anti-SPG7 (Novus NBP2-01860, 1:500), and anti-MIRO2 (Abcam ab224089, 1:1,000) were used for immunoblotting.

Techniques: Cryo-EM Sample Prep, Isolation

Journal: bioRxiv

Article Title: Structures of human organellar SPFH protein complexes

doi: 10.1101/2025.04.21.649849

Figure Lengend Snippet: a, Map-to-model fit of C-terminal PHB1 and PHB2 elements in the closed and open conformations. Note: PHB2 contains a C-terminal extension (dark blue) not present in PHB1. b, Close-up view of the hydrogen bonding network between the N-terminal portion of the CC1 helix and the β6-β7 loop in the SPFH2 domain of an adjacent subunit. c, Isolated elements contributing to the four layers at the narrow end of the PHB1/2 complex cage, colored as in . d, Side view of the cryo-EM map of the closed or open PHB1/2 complex, clipped near the center (top) shows a central channel (arrow) flanked by the PHB1 CT and the β9 ring (insets at bottom).

Article Snippet: HRP-conjugated anti-FLAG M2 (Sigma A8592, 1:5,000), HRP-conjugated StrepTactin (Bio-rad 1610381, 1:5,000), anti-Erlin1 (Proteintech 14898-1-AP, 1:1000 or Abcam ab178687, 1:10,000), anti-Erlin2 (Proteintech 16018-1-AP, 1:4,000), anti-PHB1 (Bethyl A301-873A, 1:5,000), anti-PHB2 (Bethyl A301-873A, 1:5,000), anti-AFG3L2 (Abcam ab68023, 1:500), anti-SPG7 (Novus NBP2-01860, 1:500), and anti-MIRO2 (Abcam ab224089, 1:1,000) were used for immunoblotting.

Techniques: Isolation, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structures of human organellar SPFH protein complexes

doi: 10.1101/2025.04.21.649849

Figure Lengend Snippet: a, Superposition of a PHB1/2 heterodimer in the closed (mauve and robin blue) and open (cream and gray) PHB1/2 complex conformations. b, Model of the PHB1/2 heterodimer in the closed complex conformation as in a, colored by root mean square deviation (RMSD) values compared to the open complex. Domain features are indicated. c, ‘Bottom’ view (top) of the closed and open PHB1/2 complex models with one heterodimer in each complex colored as in a and the others colored according to domain, and a rotated view showing only an isolated heterodimer (bottom). The rotation, relative to the centroid of the complex (top), and the translation (bottom) of the N-terminal residue of the PHB1 (K177, orange) or PHB2 (R191, purple) CC1 helix from the closed (mauve and robin blue) to open (cream and gray) conformation are indicated. d, Zoomed inset of the superposition as in a, showing the distance shifted by the indicated PHB1 (orange) or PHB2 (purple) residues in the SPFH domains between the closed and open conformations. e , Superposition of the isolated SPFH1 and SPFH2 domains of PHB1 and PHB2 in the closed and open PHB1/2 complex conformations, colored as in a.

Article Snippet: HRP-conjugated anti-FLAG M2 (Sigma A8592, 1:5,000), HRP-conjugated StrepTactin (Bio-rad 1610381, 1:5,000), anti-Erlin1 (Proteintech 14898-1-AP, 1:1000 or Abcam ab178687, 1:10,000), anti-Erlin2 (Proteintech 16018-1-AP, 1:4,000), anti-PHB1 (Bethyl A301-873A, 1:5,000), anti-PHB2 (Bethyl A301-873A, 1:5,000), anti-AFG3L2 (Abcam ab68023, 1:500), anti-SPG7 (Novus NBP2-01860, 1:500), and anti-MIRO2 (Abcam ab224089, 1:1,000) were used for immunoblotting.

Techniques: Cream, Isolation, Residue

Journal: bioRxiv

Article Title: Structures of human organellar SPFH protein complexes

doi: 10.1101/2025.04.21.649849

Figure Lengend Snippet: a, Surface representation of the closed (left) or open (right) PHB1/2 complex model, with two individual subunits colored as indicated, and the other subunits colored by domain. The buried area at the intersubunit interface on each side of the indicated PHB2 subunit is listed. b , Model of the N-terminal transmembrane (TM) and SPFH1 domains of PHB1 (mauve) and PHB2 (robin blue) with hydrophobic residues at the inner mitochondrial membrane (IMM) interface indicated. Aromatic residues are indicated in bold. IMS, intermembrane space. c, Model of alternating PHB1 and PHB2 subunits in the closed PHB1/2 complex with the SPFH2 domains colored as in a. Residues involved in intersubunit hydrogen bonds via their sidechain (yellow) or backbone (mauve or robin blue) are indicated. Residues involved in hydrophobic packing are orange. d, As in c, but for the open PHB1/2 complex and PHB1 colored cream. Residues indicated in bold maintain interactions in both the closed and open PHB1/2 complex. e, Model of alternating PHB1 and PHB2 CC1 helices in the closed (left) or open (right) complex, with hydrogen bonding interactions indicated as in c and d. f, Clipped side view of the cryo-EM map (left) or top view of the model (right) of the closed PHB1/2 complex with the elements that form four structural layers at the narrow end of the cage colored as indicated. g, Hydrogen bonding interactions involving the CC2 domains of PHB1 (mauve) and PHB2 (orange or robin blue), shown on the model of the closed PHB1/2 complex. The helix domain of the ‘leftmost’ PHB2 subunit, when viewed from the outside of the cage, is orange. h, Hydrophobic packing interactions involving the CC2 and CT domains of PHB1 (mauve) and PHB2 (colored according to the indicated 4-layer feature for the leftmost subunit, when viewed from the outside of the cage, or robin blue). Select residues of PHB1 (mauve), the PHB2 subunit to its left (orange), or the PHB2 subunit to its right (robin blue) are indicated.

Article Snippet: HRP-conjugated anti-FLAG M2 (Sigma A8592, 1:5,000), HRP-conjugated StrepTactin (Bio-rad 1610381, 1:5,000), anti-Erlin1 (Proteintech 14898-1-AP, 1:1000 or Abcam ab178687, 1:10,000), anti-Erlin2 (Proteintech 16018-1-AP, 1:4,000), anti-PHB1 (Bethyl A301-873A, 1:5,000), anti-PHB2 (Bethyl A301-873A, 1:5,000), anti-AFG3L2 (Abcam ab68023, 1:500), anti-SPG7 (Novus NBP2-01860, 1:500), and anti-MIRO2 (Abcam ab224089, 1:1,000) were used for immunoblotting.

Techniques: Membrane, Cream, Cryo-EM Sample Prep