The Plus End-Directed Microtubule (Kinesin-3 Family) Motor Protein KIF13B Is Associated with the Photoreceptor Synaptic Ribbon Complex
Abstract
1. Introduction
2. Results
3. Discussion
4. Materials and Methods
4.1. Materials
4.1.1. Animals
4.1.2. Antibodies
Primary Antibodies
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- anti-KIF13B rabbit polyclonal, antigen affinity-purified (commercial HPA025023, Sigma, Taufkirchen, Germany). The antigen affinity-purified antibody was raised against the peptide LTGKGKLSRRSISSPNVNRLSGSRQDLIPSYSLGSNKGRWESQQDVSQTTVSRGIAPAPA LSVSPQNNHSPDPGLSNLAASYLNP deduced from human KIF13B (amino acid (aa)1369-aa1453; Q9NQT8-1; NM_015254.4, encoding a 1826 aa long protein with a predicted mass of ≈203 kDa) [127]. This peptide stretch is highly conserved in mouse KIF13B (86% amino acid identity in comparison to the human aa sequence) and bovine KIF13B (88% amino acid identities in comparison to the human sequence). This polyclonal KIF13B antibody was used for immunofluorescence (IF) microscopy in a 1:500 dilution, for Western blot (WB) analyses in a 1:1000 dilution and for multi-peptide array WB (“Pepspots“) in a 1:100,000 dilution.
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- anti-KIF13B mouse monoclonal: (clone 5C10; IgG2b subtype) was raised against the GST tagged expressed protein of respective KIF13B encoding the sequence stretch KGKLSRRSISSPNVNRLSGSRQDLIPSYSLGSNKGRWESQQDVSQTTVSRGIAPAPALSV SPQNNHSPDPGLSNLAASYLN of mouse KIF13B (aa1371-aa1452 of NM_001081177.3 encoding a 1843 aa long protein) cloned in pGEX-6P-1. The peptide sequence is highly conserved (78% amino acid identities in bovine KIF13B, DAA27057.1; 85% amino acid identities in human KIF13B, EAW63491.1). Cloning of the GST-tagged KIF13B-encoding construct, fusion protein expression and purification, mouse immunization, generation of hybridoma cells, ELISA screening and antibody sub-typing was performed by Absea, Beijing, China. This monoclonal KIF13B antibody was used for IF and EM in a 1:100 dilution, for WB in a 1:1000 dilution and for multi-peptide array WB (“Pepspots”) in a 1:50,000 dilution.
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- Additional primary antibodies used in the present study are described in Table 1.
Secondary Antibodies
4.1.3. Plasmids
Eukaryotic KIF13B Expression Plasmid
4.2. Methods
4.2.1. Preparation of Cryostat Section and Immunolabeling of Cryostat Sections
4.2.2. Confocal- and Super-Resolution Structured-Illumination Microscopy (SR-SIM)
4.2.3. Antibody Pre-Absorption Control Experiments
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- KIF13B (Sigma, Taufkirchen, Germany)
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- KIF13B (clone 5C10)
4.2.4. Expression and Purification of Recombinant Fusion Protein
4.2.5. Post-Embedding Immunogold Labeling
4.2.6. Multi-Peptide Arrays (“Pepspots”)
4.2.7. Heterologous Protein Expression in COS7 Cells
4.2.8. SDS-PAGE and Western Blot
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Primary Antibodies | References/Source | Dilution |
---|---|---|
anti-RIBEYE mouse monoclonal antibody (2D9) against the C-terminus of RIBEYE(B)-domain/CtBP2 | [28,61] | 1:1000 (IF) 1:1000 (WB) 1:1000 (EM) |
anti-actin (clone C4) mouse monoclonal antibody | Millipore, Burlington, MA, USA, MAB1501 [128] | 1:1000 (WB) |
anti-RIBEYE rabbit polyclonal antibody (U2656) against RIBEYE(B)-domain | [23] | 1:1000 (IF) |
anti-PSD95 (post-synaptic density protein 95) rabbit polyclonal antibody (L667) | [129] | 1:1000 (IF) |
anti-mGluR6 rabbit polyclonal antibody (1205) | [26,130] | 1:2000 (IF) |
anti-Cav1.4 Cterm rabbit polyclonal antibody | [131] | 1:1000 (IF) |
Antibody | Source | Dilution |
---|---|---|
Chicken anti-mouse Alexa488 | Invitrogen Molecular Probes, Eugene, OR, USA, A-21200 | 1:1000 (IF) |
Donkey anti-mouse Alexa488 | Invitrogen Molecular Probes, A-21202 | 1:1000 (IF) |
Donkey anti-rabbit Alexa568 | Invitrogen, Molecular Probes, A-10042 | 1:1000 (IF) |
Chicken anti-rabbit Alexa488 | Invitrogen, Molecular Probes, A-21441 | 1:1000 (IF) |
Donkey anti-mouse Alexa568 | Invitrogen, Molecular Probes, A-10037 | 1:1000 (IF) |
Goat anti-mouse Alexa647 | Invitrogen, Molecular Probes, A-21236 | 1:1000 (IF) |
Donkey anti-mouse Alexa647 | Invitrogen, Molecular Probes, A-31571 | 1:1000 (IF) |
Goat anti-rabbit peroxidase-conjugated (POX) IgG | Sigma, A-6154 | 1:3000 (WB) 1:10,000 (Pepspots) |
Goat anti-mouse peroxidase-conjugated (POX) IgG | Sigma, A-3673 | 1:3000 (WB) 1:10,000 (Pepspots) |
Goat anti-mouse secondary antibody conjugated to 5 nm gold particles | Sigma, G7527 | 1:100 (EM) |
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Suiwal, S.; Schwarz, K.; Maxeiner, S.; Schmitz, F. The Plus End-Directed Microtubule (Kinesin-3 Family) Motor Protein KIF13B Is Associated with the Photoreceptor Synaptic Ribbon Complex. Int. J. Mol. Sci. 2025, 26, 6044. https://doi.org/10.3390/ijms26136044
Suiwal S, Schwarz K, Maxeiner S, Schmitz F. The Plus End-Directed Microtubule (Kinesin-3 Family) Motor Protein KIF13B Is Associated with the Photoreceptor Synaptic Ribbon Complex. International Journal of Molecular Sciences. 2025; 26(13):6044. https://doi.org/10.3390/ijms26136044
Chicago/Turabian StyleSuiwal, Shweta, Karin Schwarz, Stephan Maxeiner, and Frank Schmitz. 2025. "The Plus End-Directed Microtubule (Kinesin-3 Family) Motor Protein KIF13B Is Associated with the Photoreceptor Synaptic Ribbon Complex" International Journal of Molecular Sciences 26, no. 13: 6044. https://doi.org/10.3390/ijms26136044
APA StyleSuiwal, S., Schwarz, K., Maxeiner, S., & Schmitz, F. (2025). The Plus End-Directed Microtubule (Kinesin-3 Family) Motor Protein KIF13B Is Associated with the Photoreceptor Synaptic Ribbon Complex. International Journal of Molecular Sciences, 26(13), 6044. https://doi.org/10.3390/ijms26136044