anti-mouse β-actin # 5125  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Proximity Labeling of NIMA Kinase Complex Components in C. elegans

doi: 10.1101/2025.06.16.659960

Figure Lengend Snippet: (A) Schematic of the two NEKL–MLT protein subcomplexes (i.e., MLT-4–MLT-2–NEKL-2 and MLT-3–NEKL-3); dashed lines indicate putative contacts between the subcomplexes. (B) AlphaFold3 modeling and ChimeraX rendering of predicted MLT-4–MLT-2–NEKL-2 and MLT-3–NEKL-3 subcomplexes. Proteins are represented as backbone ribbon diagrams with partially transparent surfaces. Black lines between proteins indicate high-confidence Predicted Aligned Error (PAE) pseudobonds (PAE ≤ 6Å; interatomic-distance ≤ 5Å). For clarity, highly unstructured portions of the peptides (B-factor ≤ 30) were hidden. For additional information see . (D) Diagram of the proximity labeling experimental steps including schematic of a C-terminally tagged bait protein containing an unstructured linker, TurboID, and 3xFLAG-tag. Proteins within the red shaded area, indicated by colored shapes, are biotinylated by the bait–TurboID fusion in vivo. After lysis, biotinylated proteins are captured and enriched on streptavidin beads via washing, followed by trypsin digestion. Released peptide fragments are identified by LC-MS/MS, followed by filtering and annotation of the datasets. (D–F) Western blot analyses of input lysates and purified eluates (after enrichment on beads) from the indicated strains to detect biotinylated proteins (streptavidin), bait–TurboID fusion proteins (FLAG), and β-actin. Arrowheads in western blots are color-coded to correspond to the NEKL–MLT bait proteins in panel A. Red triangles correspond to the major biotinylated carboxylase species; green triangles correspond to the P dpy-7 ::mNG::TurboID control. (D) Lysates of N2, NEKL-2::TurboID, and NEKL-3::TurboID showing faint biotinylated protein species in the bait–TurboID lanes. (E) Lysates of N2 and NEKL-3::TurboID showing the strong depletion of biotinylated proteins within one hour of incubation with streptavidin beads. E’ shows quantification of the indicated bands from panel F. Also note the progressive depletion of NEKL-3::TurboID (FLAG) from the lysate. (F). Representative TurboID experimental blot (Experiment 6, Replicate 3) showing input lysates and purified eluates. Note differences in the levels of the bait–TurboID proteins as well as the strongly expressed P dpy-7 ::mNG::TurboID control. Full blots are available in S1 Data.

Article Snippet: Antibodies used in this study were Streptavidin-HRP (Cell Signaling Technology, Cat# 3999S), β-Actin (13E5)-HRP (Cell Signaling Technology, Cat# 5125S), and THE DYKDDDDK [FLAG-TAG] Antibody-HRP (GenScript, Cat# A01428).

Techniques: Labeling, In Vivo, Lysis, Liquid Chromatography with Mass Spectroscopy, Western Blot, Purification, Control, Incubation

Journal: bioRxiv

Article Title: Proximity Labeling of NIMA Kinase Complex Components in C. elegans

doi: 10.1101/2025.06.16.659960

Figure Lengend Snippet: (A, C, D) Proportional Venn diagrams showing the extent of overlap in the proteins detected by technical replicates from the indicated strains in (A) Exp 1 and 2, (C) Exp 4, and (D) Exp 7; n indicates the total number of unique proteins. Color key for C and D is shown in A. (B–D) Scatter plots showing correlations in protein abundance (intensities) between replicate pairs from the indicated experiments; dots correspond to individual proteins detected in both replicates. R 2 values were calculated using simple linear regression; raw data are available in S11 File. (B) Scatter plots of technical replicates from N2, NEKL-2::TurboID, NEKL-3::TurboID, and MLT-3::TurboID of Exp 1. (C) (left) Overlaps for technical replicates from N2 and MLT-2::TurboID of Exp 4. (center) Scatter plots of MLT-2::TurboID (Exp 4) technical replicates. (right) Western blots corresponding to Exp 4, technical replicate 2 showing biotinylation (streptavidin; eluate), bait proteins (FLAG; eluate), and loading control (β-actin; lysate). Colored arrowheads correspond to MLT-4::TurboID (pink), MLT-2::TurboID (yellow), and the major carboxylase species (red). Note that MLT-2::TurboID is expressed at levels close to the limits of detection. (D) (left) Overlaps for technical replicates from N2 and NEKL-3::TurboID Exp 7. Note relative lack of overlap for NEKL-3::TurboID due to reduced numbers of proteins identified in replicates 1 and 2. (center) Scatter plots of NEKL-3::TurboID (Exp 4) technical replicates. (right) Western blots corresponding to Exp 7, technical replicate 2. Colored arrowheads correspond to NEKL-2::TurboID (teal), NEKL-3::TurboID (purple), MLT-3::TurboID (orange), and the major carboxylase species (red). Note reduced levels of signal in the NEKL-3 eluate.

Article Snippet: Antibodies used in this study were Streptavidin-HRP (Cell Signaling Technology, Cat# 3999S), β-Actin (13E5)-HRP (Cell Signaling Technology, Cat# 5125S), and THE DYKDDDDK [FLAG-TAG] Antibody-HRP (GenScript, Cat# A01428).

Techniques: Quantitative Proteomics, Western Blot, Control

Journal: bioRxiv

Article Title: Proximity Labeling of NIMA Kinase Complex Components in C. elegans

doi: 10.1101/2025.06.16.659960

Figure Lengend Snippet: (A–C) Western blots with specific proteins indicated by the key. (A) Streptavidin-probed western blot showing a reduction in the major carboxylase band in strains that underwent carboxylase depletion (AX7884, NEKL-2, and NEKL-3). –His, lysates were incubated with nickel-coated (His-binding) beads to remove the endogenous His-tagged versions of PYC-1, PCCA-1, MCCC-1, and POD-2. N2 does not encode His-tagged carboxylases, accounting for the stronger band in this strain. Increased protein concentration in the eluate lanes facilitates the visualization of the bait-induced biotinylated bands. (B) Western blots from Exp 3, replicate 1Exp showing biotinylated products (streptavidin), bait proteins (FLAG), and loading control (β-actin). (C) Western blot of lysates from the indicated strains after incubation in the presence or absence of auxin, which leads to the degradation of the AID-tagged proteins. Note that the NEKL-2::AID strain contained a defective FLAG tag and was thus not detectable.

Article Snippet: Antibodies used in this study were Streptavidin-HRP (Cell Signaling Technology, Cat# 3999S), β-Actin (13E5)-HRP (Cell Signaling Technology, Cat# 5125S), and THE DYKDDDDK [FLAG-TAG] Antibody-HRP (GenScript, Cat# A01428).

Techniques: Western Blot, Incubation, Binding Assay, Protein Concentration, Control, FLAG-tag