anti caveolin 1 antibody  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Control of myogenesis by the E3 ubiquitin ligase CUL3-BTBD9

doi: 10.1101/2025.05.15.654347

Figure Lengend Snippet: A. Upper panel: Domain structure of BTBD9 (DiD: discoidin domain). Lower panel: AlphaFold model of BTBD9. B. Data-independent acquisition mass spectrometry analysis of affinity-purifications of wildtype BTBD9 (x-axis) and BTBD9 ΔCUL3 (y-axis). C. FLAG BTBD9 or FLAG BTBD9 ΔCUL3 were affinity-purified from C2C12 myoblasts and endogenous co-precipitating CAV1 was detected by Western blotting. D. FLAG BTBD9 was co-expressed in C2C12 myoblasts with indicated HA-tagged proteins (‘prey’), and co-purifying proteins were detected by Western blotting. E. Control C2C12 myoblasts of BTBD9::FLAG myoblasts (endogenous BTBD9 loci were tagged with FLAG) were subjected to αFLAG affinity-purification, and co-precipitating endogenous proteins were detected by Western blotting.

Article Snippet: Depending on the experiment, cells were stained with the primary antibody anti-myosin heavy chain (DHSB; MF20) at 1:50, anti-myogenin (DHSB; F5G) at 1:50, or anti-CAV1 (Cell Signaling, 3267S) at 1:200 for 3h.

Techniques: Data-independent acquisition, Mass Spectrometry, Affinity Purification, Western Blot, Control

Journal: bioRxiv

Article Title: Control of myogenesis by the E3 ubiquitin ligase CUL3-BTBD9

doi: 10.1101/2025.05.15.654347

Figure Lengend Snippet: A. C2C12 myoblasts were treated with control siRNAs or siRNAs targeting BTBD9, CAV1, or both. Cells were transferred to differentiation medium, and myotube formation was followed by immunofluorescence microscopy against MyHC. Quantification of three independent experiments is shown on the right. B. Sequential affinity-purification of BTBD9 and CAV1 compared to single BTBD9 immunoprecipitation, as analyzed by mass spectrometry. KCTD10, Cavin1, and CUL3 are found in both purifications, while many components of the PI3K pathways are only found in BTBD9-purifications.

Article Snippet: Depending on the experiment, cells were stained with the primary antibody anti-myosin heavy chain (DHSB; MF20) at 1:50, anti-myogenin (DHSB; F5G) at 1:50, or anti-CAV1 (Cell Signaling, 3267S) at 1:200 for 3h.

Techniques: Control, Immunofluorescence, Microscopy, Affinity Purification, Immunoprecipitation, Mass Spectrometry

Journal: bioRxiv

Article Title: Control of myogenesis by the E3 ubiquitin ligase CUL3-BTBD9

doi: 10.1101/2025.05.15.654347

Figure Lengend Snippet: A. FLAG-tagged wild-type BTBD9 or variants with a mutant CUL3-binding interface (ΔCUL3) or with a deletion removing the second discoidin domain (ΔDiD2) were co-expressed with HA CAV1. Following α-FLAG affinity-purification, co-precipitating proteins were detected by Western blotting. B. AlphaFold model (iPTM 0.72) of a complex between a thioredoxin-like domain in KCTD10 and the second discoidin domain in BTBD9. C. BTBD9 variants with indicated mutations were affinity-purified, and co-precipitating HA KCTD10 was detected by Western blotting. D. Mass spectrometry analyses of immunoprecipitated BTBD9 or BTBD9 with mutations in three critical residues required for substrate engagement (BTBD9 DiD2-M ). E. The oligomerization and scaffolding domain of CAV1 is required for recognition by BTBD9. FLAG-tagged CAV1 variants were affinity-purified and co-precipitating BTBD9 was detected by Western. F. The second discoidin domain of BTBD9 is required for CAV1 recognition. The indicated BTBD9 variants were affinity-purified from C2C12 myoblasts, and co-precipitating proteins were detected by Western blotting.

Article Snippet: Depending on the experiment, cells were stained with the primary antibody anti-myosin heavy chain (DHSB; MF20) at 1:50, anti-myogenin (DHSB; F5G) at 1:50, or anti-CAV1 (Cell Signaling, 3267S) at 1:200 for 3h.

Techniques: Mutagenesis, Binding Assay, Affinity Purification, Western Blot, Mass Spectrometry, Immunoprecipitation, Scaffolding

Journal: bioRxiv

Article Title: Control of myogenesis by the E3 ubiquitin ligase CUL3-BTBD9

doi: 10.1101/2025.05.15.654347

Figure Lengend Snippet: A. Indicated FLAG BTBD9 variants were affinity-purified and co-precipitating CAV1 was detected by Western blotting. Only affinity-purification of WT-BTBD9, but not a mutant defective in catalyzing ubiquitylation (ΔCUL3) resulted in co-precipitation of a modified version of CAV1, with a molecular weight expected for monoubiquitylated CAV1. B. Denaturing NiNTA-purification of His-ubiquitin conjugates from either WT or ΔBTBD9 myoblasts shows that overexpression of BTBD9 resulted in modification of CAV1 with up to three ubiquitin molecules, as detected by Western blotting. C. Denaturing NiNTA-purification of His-ubiquitin conjugates from cells expressing single-Lys variants of CAV1 reveals that BTBD9 preferentially promotes modification of K26, K30, and K39 in CAV1 with short oligomers containing up to three ubiquitin subunits. D. Degradation reporter assay, as described , shows that deletion of BTBD9 did not alter the stability of CAV1.

Article Snippet: Depending on the experiment, cells were stained with the primary antibody anti-myosin heavy chain (DHSB; MF20) at 1:50, anti-myogenin (DHSB; F5G) at 1:50, or anti-CAV1 (Cell Signaling, 3267S) at 1:200 for 3h.

Techniques: Affinity Purification, Western Blot, Mutagenesis, Modification, Molecular Weight, Purification, Ubiquitin Proteomics, Over Expression, Expressing, Reporter Assay

Journal: bioRxiv

Article Title: Control of myogenesis by the E3 ubiquitin ligase CUL3-BTBD9

doi: 10.1101/2025.05.15.654347

Figure Lengend Snippet: A. Denaturing NiNTA-purification of His-ubiquitin conjugates from either WT or ΔBTBD9 myoblasts shows that overexpression of BTBD9 resulted in modification of CAV1 with up to three ubiquitin molecules, as detected by Western blotting. As indicated, KCTD10 was expressed either by itself or in combination with BTBD9. B. Denaturing NiNTA-purification of His-ubiquitin conjugates from either WT or ΔBTBD9 myoblasts showing that ubiquitylation of KCTD10 occurs independently of whether BTBD9 can bind CUL3 or substrate, as detected by Western blotting.

Article Snippet: Depending on the experiment, cells were stained with the primary antibody anti-myosin heavy chain (DHSB; MF20) at 1:50, anti-myogenin (DHSB; F5G) at 1:50, or anti-CAV1 (Cell Signaling, 3267S) at 1:200 for 3h.

Techniques: Purification, Ubiquitin Proteomics, Over Expression, Modification, Western Blot

Journal: bioRxiv

Article Title: Control of myogenesis by the E3 ubiquitin ligase CUL3-BTBD9

doi: 10.1101/2025.05.15.654347

Figure Lengend Snippet: A. Mass spectrometry analyses of immunoprecipitated FLAG CAV1 from either WT or ΔBTBD9 myoblasts. Known CAV1 interactors are shown in red. B. WT or ΔBTBD9 C2C12 myoblasts were transferred to differentiation medium, and the indicated proteins were detected over time by Western blotting. C. WT or ΔBTBD9 C2C12 myoblasts were analyzed for CAV1 expression by immunofluorescence microscopy against the endogenous proteins. Quantification of three independent experiments is shown on the right. D. Analysis of expression of CAV1 mRNA using qRT-PCR, in WT or ΔBTBD9 C2C12 myoblasts either prior to initiation of differentiation (d0) or two days after initiation of differentiation (d2). E. Analysis of expression of various FOXO target genes using qRT-PCR, in WT or ΔBTBD9 C2C12 myoblasts either prior to initiation of differentiation (d0) or two days after initiation of differentiation (d2). F. WT or ΔBTBD9 C2C12 myoblasts were transferred to differentiation medium, and the indicated components of the PI3K-AKT signaling pathway were detected over time by Western blotting. G. The indicated C2C12 myoblasts lines were subjected to brief serum starvation and re-stimulated with insulin. Protein expression was analyzed by Western blotting.

Article Snippet: Depending on the experiment, cells were stained with the primary antibody anti-myosin heavy chain (DHSB; MF20) at 1:50, anti-myogenin (DHSB; F5G) at 1:50, or anti-CAV1 (Cell Signaling, 3267S) at 1:200 for 3h.

Techniques: Mass Spectrometry, Immunoprecipitation, Western Blot, Expressing, Immunofluorescence, Microscopy, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Control of myogenesis by the E3 ubiquitin ligase CUL3-BTBD9

doi: 10.1101/2025.05.15.654347

Figure Lengend Snippet: A. Localization of endogenous BTBD9 and IGF1-R was analyzed in WT C2C12 myoblasts, ΔCAV1 myoblasts, or WT myoblasts treated with the CUL3 inhibitor MLN4924. A larger magnification of the insert focusing on plasma membrane-localized proteins is shown below. B. Affinity-purification of endogenously FLAG-tagged BTBD9 from either wildtype or ΔCAV1 myoblasts. Co-precipitating proteins were detected by Western blotting. C. Competition assay between wildtype C2C12 myoblasts stably expressing GFP and ΔBTBD9 myoblasts stably expressing mCherry. Cells were mixed at a 1:1 ratio, briefly serum-starved, and restimulated with either insulin or IGF1. GFP/mCherry-positive cells were detected 24h later by flow cytometry. D. Competition assay between wildtype C2C12 myoblasts stably expressing GFP and ΔCAV1 myoblasts stably expressing mCherry. Cells were mixed at a 1:1 ratio, briefly serum-starved, and restimulated with either insulin or IGF1. GFP/mCherry-positive cells were detected 24h later by flow cytometry.

Article Snippet: Depending on the experiment, cells were stained with the primary antibody anti-myosin heavy chain (DHSB; MF20) at 1:50, anti-myogenin (DHSB; F5G) at 1:50, or anti-CAV1 (Cell Signaling, 3267S) at 1:200 for 3h.

Techniques: Clinical Proteomics, Membrane, Affinity Purification, Western Blot, Competitive Binding Assay, Stable Transfection, Expressing, Flow Cytometry