rat anti alpha tubulin  (Bio-Rad)

Journal: bioRxiv

Article Title: Mammalian metaphase kinetochores are elastic and require condensin for robust structure and function

doi: 10.64898/2025.12.23.696255

Figure Lengend Snippet: (A) Representative PtK2 SMC2 RNAi cell timelapse of eGFP-CENP-A deforming with the microneedle pulling, and relaxing. Yellow box shows the zoomed in pair on the right, cyan arrow shows direction of needle movement, white arrowheads highlight the “front” kinetochore, and grey arrowheads highlight the “back” kinetochore. (B) K-K distance over time before, during, and after microneedle pulling for n = 9 kinetochores. “Initial” length is the frame just before pulling begins. Time = 0 s corresponds to the time maximum K-K distance was reached and measured during pulling, highlighting relaxation dynamics of each pulled kinetochore. The grey box indicates the pulling period. (C) CENP-A lengths over time before, during, and after microneedle pulling for 9 kinetochores as defined in (B). (D) CENP-A length at the frame before microneedle pulling, at the maximum K-K distance during pulling, and 30 s after pulling in siSMC2 cells (n = 9 kinetochores; Paired t-test). (E) Percent of K-K distance and CENP-A length increase from the frame before pulling begins to maximum measured K-K distance (n = 9 kinetochores; Paired t-test). (F) eGFP-CENP-A images at maximum measured K-K distance and 30s afterwards of control deformation and two siSMC2 kinetochore deformations exhibiting dramatic “tails”. (G) Percentage of control (n = 6) and siSMC2 (n = 9) kinetochore pulls with persistent “tails” for > 30 s during the relaxation period. (H) Timelapse comparing representative K-K distance relaxation for a control pull and for a fast relaxation SMC2 RNAi pull during the needle hold. White box indicates zoomed in pair. Grey dashed lines project the kinetochores’ movements over time. (I) Examples of non-detached (15 s after maximum K-K) and detached kinetochore (10 s after maximum K-K) from siSMC2 pulling experiments with fast K-K relaxation rates. Inset is of the front kinetochore with a linescan for tubulin intensity and kinetochore intensity; corresponding plots on the right annotated with sections of intensity signal corresponding to k-fiber presence or not. (J) K-K distance change after maximum K-K distance (t = 0) for control (black, n = 6 kinetochores), and detached, siSMC2 kinetochores (red and brown line, n = 2/9 kinetochores) pulls. (K) Percentage of pulls that led to detachment events in control and siSMC2 cells based on two criteria: fast K-K distance relaxation as in (J) and loss of tubulin signal attached to the kinetochore as in (I).

Article Snippet: The following primary antibodies were used: mouse anti-Hec1 (1:1000; Novus Biologicals; NB100-338), chicken anti-GFP (1:500; Aves Lab Inc.; GFP-1010), rabbit anti-alpha tubulin (1:500; Abcam; ab18251), rat anti-alpha tubulin (1:2000; Bio-Rad; REFMCA77G).

Techniques: Control

Journal: The Journal of Biological Chemistry

Article Title: Hsp90 co-chaperone FKBP4 facilitates CCT8 folding and connects Hsp90 to chaperonin-dependent proteostasis

doi: 10.1016/j.jbc.2025.110914

Figure Lengend Snippet: FKBP4 prevents CCT8 aggregation. A , following the knockdown of FKBP4 in HCT116 cells for 72 h, the aggregation of CCT1 and CCT8 proteins within cells was assessed using a filter trap assay. Cell lysates were filtered through a nitrocellulose membrane, and the retained proteins on the membrane were detected using specific antibodies against CCT1 and CCT8. B , quantitative analysis of CCT1 and CCT8 protein aggregation, normalized to GAPDH, in shFKBP4 HCT116 cells. C , cell lysates that did not pass through the nitrocellulose membrane were subjected to western blotting to detect the levels of FKBP4, CCT8, CCT1, CDK2, α-tubulin, and GAPDH within the cells. D , quantitative analysis of CCT8 and CCT1 protein expression, normalized to GAPDH, in shFKBP4 HCT116 cells. E , following the knockdown of FKBP4 in HCT116 cells for 72 h, the aggregation of CCT2 and CCT3 proteins within cells was assessed using a filter trap assay. The experimental procedure mirrored that of ( A ). F , quantitative analysis of CCT2 and CCT3 protein aggregation, normalized to GAPDH, in shFKBP4 HCT116 cells. G , cell lysates that did not pass through the nitrocellulose membrane were subjected to western blotting to detect the levels of FKBP4, CCT2, CCT3, and GAPDH within the cells. The experimental procedure mirrored that of ( C ). H , quantitative analysis of CCT2 and CCT3 protein expression, normalized to GAPDH, in shFKBP4 HCT116 cells. I , shFKBP4 HCT116 cells were harvested after 3 days of puromycin selection. The cell lysates were biochemically fractionated into Triton-soluble and -insoluble fractions as described in “ ”. The FKBP4 expression levels and Triton-soluble and -insoluble fractions of CCT8 were analyzed by western blotting. The GAPDH was used as a loading control. J , The ratio of the CCT8 Triton-insoluble form in ( I ). K , The primary structures of FKBP4 and FKBP5. L , after 48 h of transfection, vector, FLAG-tagged FKBP4 or FLAG-tagged FKBP5 expressing HEK293T cell lysates were precipitated by anti-FLAG antibodies, and the products were detected for the co-purification of the endogenous proteins. M , comparison of the interaction between Hsp90 and FLAG-tagged FKBP4 or FLAG-tagged FKBP5 in ( L ). N , following the knockdown of FKBP5 in HCT116 cells for 72 h, the aggregation of CCT8 proteins within cells was assessed using a filter trap assay. The experimental procedure mirrored that of ( A ). O , quantitative analysis of the protein aggregation of CCT8, normalized to GAPDH, in shFKBP5 HCT116 cells. P , cell lysates that did not pass through the nitrocellulose membrane were subjected to western blotting to detect the levels of FKBP5, CCT8, and GAPDH within the cells. The experimental procedure mirrored that of ( C ). Q , quantitative analysis of CCT8 protein expression, normalized to GAPDH, in shFKBP5 HCT116 cells. R , shFKBP5 HCT116 cells were harvested after puromycin selection for 3 days. The experimental procedure mirrored that of ( I ). S , the ratio of the CCT8 Triton-insoluble form in ( R ). T , HCT116 cells were treated with 17-AAG (20 μM) or ganetespib (2 μM) for 24 h, and CCT8 aggregation was assessed by filter trap assay. U , quantitative analysis of CCT8 protein aggregation with GAPDH serving as an internal control in ( T ). V , cell lysates that did not pass through the nitrocellulose membrane were subjected to western blotting to detect the levels of HSPA1A, CCT8, HSP90, and GAPDH within the cells. The experimental procedure mirrored that of ( C ). W , quantitative analysis of CCT8 protein expression was normalized with GAPDH in ( V ). All data were analyzed with an unpaired two-tailed Student's t test. Each dataset is expressed as mean ± SD for n = 3. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Primary antibodies were used to detect FKBP4 (1:1000, ab97306, Abcam), GAPDH (1:1000, GTX100118, GeneTex, USA), E-cadherin (1:1000, 610182, BD), FASN (1:1000, GTX109833, GeneTex), FLNA (1:1000, GTX112939, GeneTex), TALIN-1 (1:500, GTX102215, GeneTex), MRE11 (1:1000, PC388, Calbiochem), DDX3 (1:1000, GTX110614, GeneTex), TCP1 theta (CCT8) (1:1000, GTX105725, GeneTex), CDK2 (1:500, 2546, Cell Signaling), CCT1 (1:250, sc-53454, Santa-cruz Biotechnology), HSP90 (1:1000, sc-13119, Santa-cruz Biotechnology), HSPA1A (1:1000, sc-66048, Santa-cruz Biotechnology), FKBP5 (1:1000, GTX113438, GeneTex), α-tubulin (1:1000, MCA78G, Bio-Rad Laboratories Inc), FLAG (1:2000, F3165, Sigma-Aldrich), c-Myc (1:1000, #11667203001, Roche), CCT2 (1:500, sc-374152, Santa-cruz Biotechnology), CCT3 (1:500, sc-271336, Santa-cruz Biotechnology), HA (1:5000, MMS-101R, Covance), LexA (1:1000, sc-7544, Santa-cruz Biotechnology), and Pgk1 (1:10000, 459250, Invitrogen).

Techniques: Knockdown, TRAP Assay, Membrane, Western Blot, Expressing, Selection, Control, Transfection, Plasmid Preparation, Copurification, Comparison, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Hsp90 co-chaperone FKBP4 facilitates CCT8 folding and connects Hsp90 to chaperonin-dependent proteostasis

doi: 10.1016/j.jbc.2025.110914

Figure Lengend Snippet: The model of the Hsp90-FKBP4 folding cycle. FKBP4 binds to Hsp90 through the C-terminal TRP motif, forming an Hsp90-FKBP4 protein complex to execute protein folding. FKBP4 can help fold CCT8, one of the TRiC subunits. CCT8, when well folded by the Hsp90-FKBP4 complex, can form a tubular chaperonin structure with other TRiC subunits. Functional chaperonin is known to be responsible for the folding of α-tubulin.

Article Snippet: Primary antibodies were used to detect FKBP4 (1:1000, ab97306, Abcam), GAPDH (1:1000, GTX100118, GeneTex, USA), E-cadherin (1:1000, 610182, BD), FASN (1:1000, GTX109833, GeneTex), FLNA (1:1000, GTX112939, GeneTex), TALIN-1 (1:500, GTX102215, GeneTex), MRE11 (1:1000, PC388, Calbiochem), DDX3 (1:1000, GTX110614, GeneTex), TCP1 theta (CCT8) (1:1000, GTX105725, GeneTex), CDK2 (1:500, 2546, Cell Signaling), CCT1 (1:250, sc-53454, Santa-cruz Biotechnology), HSP90 (1:1000, sc-13119, Santa-cruz Biotechnology), HSPA1A (1:1000, sc-66048, Santa-cruz Biotechnology), FKBP5 (1:1000, GTX113438, GeneTex), α-tubulin (1:1000, MCA78G, Bio-Rad Laboratories Inc), FLAG (1:2000, F3165, Sigma-Aldrich), c-Myc (1:1000, #11667203001, Roche), CCT2 (1:500, sc-374152, Santa-cruz Biotechnology), CCT3 (1:500, sc-271336, Santa-cruz Biotechnology), HA (1:5000, MMS-101R, Covance), LexA (1:1000, sc-7544, Santa-cruz Biotechnology), and Pgk1 (1:10000, 459250, Invitrogen).

Techniques: Functional Assay

Journal: bioRxiv

Article Title: Structure and function of the nervous system in the stem of the siphonophore Nanomia septata : its role in swimming coordination

doi: 10.1101/2025.11.27.690755

Figure Lengend Snippet: (A), colony anterior showing pneumatophore (pn), nectophores and gastrozooids (gz) attached to the central stem, visible through the transparent nectosome. (B), entire isolated nectophore stained with anti-tubulin antibody (green) and phalliodin (red); both the upper nerve (un) and the lower nerve (ln) join the nerve ring (rn); endodermal muscles (em); the terminal ganglion (tg) is located at the back of the nectophore where it normally contacts the stem. (C), the lower nerve (ln) terminates in the terminal ganglion (tg). (D), the point at which the lower nerve (ln) joins the nerve ring (rn) shown by an arrow. (E), terminal ganglion (tg) at high magnification; nuclei (blue) shown by DAPI labeling. Scale bars: A, 5 mm; B, 500 µm; C, D, 200 µm; E, 30 µm;

Article Snippet: Therefore, as a first marker, rat monoclonal anti-tubulin antibody was used (AbD Serotec, Bio-Rad, Cat# MCA77G, RRID: AB_325003), which recognizes the alpha subunit of α-tubulin, specifically binding tyrosylated α-tubulin ( ; ).

Techniques: Isolation, Staining, Muscles, Labeling

Journal: bioRxiv

Article Title: Structure and function of the nervous system in the stem of the siphonophore Nanomia septata : its role in swimming coordination

doi: 10.1101/2025.11.27.690755

Figure Lengend Snippet: Stem nervous system labeled with anti-tubulin antibody (green); nuclear DAPI staining (blue) . (A), stem anterior where it connects to the pneumatophore (pn) showing the origin of the giant axon (ga); the nerve network that covers the outer layer of the pneumatophore merges into the stem. (B), giant axon (ga) running along the entire stem. (C, D), the polygonal nerve network covers the subepithelial layer in the stem and merges with the giant axons (ga) at numerous points (arrows). (E), the cone-shaped structure, which serves as a nectophore docking site, contains the same polygonal nerve network as the stem. (F), higher magnification of the cone and its polygonal nerve network; arrows point to the nectophore attachment surface; cell nuclei (blue). Scale bars: A, B, 200 µm; C–F, 100 µm.

Article Snippet: Therefore, as a first marker, rat monoclonal anti-tubulin antibody was used (AbD Serotec, Bio-Rad, Cat# MCA77G, RRID: AB_325003), which recognizes the alpha subunit of α-tubulin, specifically binding tyrosylated α-tubulin ( ; ).

Techniques: Labeling, Staining

Journal: bioRxiv

Article Title: Structure and function of the nervous system in the stem of the siphonophore Nanomia septata : its role in swimming coordination

doi: 10.1101/2025.11.27.690755

Figure Lengend Snippet: (Ai), two lateral giant axons (ga) and their associated nerve network (green), together with FMRFa-ir neural tracts connecting contralateral cones. (Aii), red channel of (Ai) showing FMRFa IR only; each neural tract connects contralateral cones (arrows) but not immediate neighbours. (Aiii), green channel of (Ai) showing tubulin IR only. (B), higher power of the stem showing the giant axons (ga), the polygonal nerve network (green), the neural tract (red; arrows), all running along the stem; there is a neural loop at the tip of each cone. (Ci), cone with its polygonal nerve network (green) and FMRFa-ir neural loop (red) at the cone tip. (Cii), red channel of (Ci) showing FMRFa-ir labeling only; arrows show some of the numerous neural cell bodies. (Di), high magnification of the polygonal nerve network (green) and the FMRFa-ir neural tract (red). (Dii), red channel of (Di) showing FMRFa IR only; arrows show some of the immunoreactive cell bodies. (Diii), green channel of (Di) showing that the FMRFa-ir neural tract does not label with tubulin IR. Scale bars: Ai-Aiii, 500 µm; B, 200 µm; Ci, Cii, 100 µm; Di-Diii, 50 µm.

Article Snippet: Therefore, as a first marker, rat monoclonal anti-tubulin antibody was used (AbD Serotec, Bio-Rad, Cat# MCA77G, RRID: AB_325003), which recognizes the alpha subunit of α-tubulin, specifically binding tyrosylated α-tubulin ( ; ).

Techniques: Labeling

Journal: bioRxiv

Article Title: Structure and function of the nervous system in the stem of the siphonophore Nanomia septata : its role in swimming coordination

doi: 10.1101/2025.11.27.690755

Figure Lengend Snippet: (Ai), double labeled image. (Aii), red channel with FMRFa IR only. (Aiii), green channel with tubulin IR only; yellow arrow indicates a thick process with only tubulin IR; white arrows indicate processes with cross-reactivity for both tubulin IR and FMRFa IR. (Bi), double labeled image of the stem with giant axon (ga) and adjacent nerve network; asterisk shows pentagonal shaped neural unit. (Bii), red channel, with FMRFa-ir processes (arrows) that cross the giant axon rather than merging with it. (Biii), green channel showing that the giant axon (ga) is labeled only with tubulin IR; yellow arrow shows tubulin-ir threads merging with the giant axon. (Ci) pentagonal shaped neural unit from Bi; (Cii) red channel with FMRFa IR only; (Ciii) green channel with tubulin IR only. Yellow arrows indicate thick neural threads with tubulin IR only; white arrows indicate processes with both tubulin IR and FMRFa IR. Scale bars: A, B, 50 µm; C, 12 µm.

Article Snippet: Therefore, as a first marker, rat monoclonal anti-tubulin antibody was used (AbD Serotec, Bio-Rad, Cat# MCA77G, RRID: AB_325003), which recognizes the alpha subunit of α-tubulin, specifically binding tyrosylated α-tubulin ( ; ).

Techniques: Labeling

Journal: bioRxiv

Article Title: Structure and function of the nervous system in the stem of the siphonophore Nanomia septata : its role in swimming coordination

doi: 10.1101/2025.11.27.690755

Figure Lengend Snippet: (A), the stem neural system includes two giant axons, two tubulin-ir nerve networks and a system of FMRFa-ir, double-threaded, neural tracts between contralateral cones; the stem itself consists of two columns of cone-shaped protrusions that serve as docking stations for the nectophores; the terminal ganglion is located at the attachment point; it is connected to the nerve ring, in the margin of the nectophore, by the lower nerve; also shown are the Claus muscles of the velum (cm) and the muscles of the endoderm (em) that abut onto them . (B), detailed view of the cone attachment area between the nectophore and the stem; the terminal ganglion of the nectophore is separated from the cone surface by a simple narrow cleft. Outside of the terminal ganglion contact, the epithelial layer of the nectophore is firmly attached to the cone. In this area (black dotted line), electrical junctions may couple the nerve network of the cone with the epithelial conductance pathway in the nectophore.

Article Snippet: Therefore, as a first marker, rat monoclonal anti-tubulin antibody was used (AbD Serotec, Bio-Rad, Cat# MCA77G, RRID: AB_325003), which recognizes the alpha subunit of α-tubulin, specifically binding tyrosylated α-tubulin ( ; ).

Techniques: Muscles

Journal: bioRxiv

Article Title: Structure and function of the nervous system in the stem of the siphonophore Nanomia septata : its role in swimming coordination

doi: 10.1101/2025.11.27.690755

Figure Lengend Snippet: (A), early stage nectophore attached to the stem by an extended side-branch (sb). (B), later stage nectophore showing lower nerve (ln) and terminal ganglion (tg) at the point of contact with the shortened stem branch (sb); arrow indicates the FMRFa-ir neural loop (red). (C), mature nectophore connected to the stem via a short cone. (Di), higher magnification of the terminal ganglion (tg) area from (C); the terminal ganglion and cone are separated by a narrow cleft (arrow); many thin nerve fibres, both tubulin-ir and FMRFa-ir, come to the cone surface here. (Dii), green channel of (Di) showing the nerve fibres opposite the nectophore terminal ganglion (tg). (Diii), red channel of (Di) showing FMRFa-ir cell bodies; labeling absent from terminal ganglion or lower nerve. (E), side view of the contact between the terminal ganglion and the cone, showing the thin nerve fibres at the cone surface, opposite the narrow cleft (arrows) that separates the cone from the terminal ganglion; nerve contains both tubulin-ir and FMRFa-ir elements. (F), different view of the terminal ganglion (tg), from the side and slightly above, showing that the polygonal nerve network gives rise to numerous fine processes (arrows) that project toward the terminal ganglion. (G), high magnification of (C) showing the area of contact between the nectophore epithelium and the cone surface (arrow); the outline of the epithelial cells is revealed by background tubulin-ir. D, E and F, different preparations. Scale bars: A, B, 200 µm; C, 100 µm; Di-Diii, G, 50 µm; E, 40 µm; F, 30 µm.

Article Snippet: Therefore, as a first marker, rat monoclonal anti-tubulin antibody was used (AbD Serotec, Bio-Rad, Cat# MCA77G, RRID: AB_325003), which recognizes the alpha subunit of α-tubulin, specifically binding tyrosylated α-tubulin ( ; ).

Techniques: Labeling