rabbit polyclonal anti kifc3  (Proteintech)

Journal: Cell Communication and Signaling : CCS

Article Title: Kinesin KIFC3 is essential for microtubule stability and cytokinesis in oocyte meiosis

doi: 10.1186/s12964-024-01589-8

Figure Lengend Snippet: KIFC3 is essential for mouse oocyte meiotic maturation ( A ) Oocytes were immunolabeled with anti-α-tubulin (green) and anti-KIFC3 antibody (red), and Hoechst 33,342 was used to label DNA (blue). Bar = 20 μm. ( B ) Co-localization of KifC3 with Crest at MI and MII stages. Oocytes cultured to 8 h (MI) and 12 h (MII) were stained for KIFC3 (green), Crest (red), and DNA (blue). Bar = 10 μm. ( C ) Immunoblotting assays for the expression level of KIFC3 at different stages (GV, GVBD, MI, and MII) during mouse oocyte maturation. ( D ) The typical picture of control oocytes and KIFC3-antibody injection oocytes. Bar = 50 μm. ( E ) Quantitative analysis of GVBD rate and PB1 extrusion rate in control and KIFC3-antibody injection oocytes. Graph shows means ± sem of results obtained in 3 independent experiments. * P < 0.05

Article Snippet: The rabbit polyclonal anti-KIFC3 antibody (10125-2-AP) and rabbit polyclonal anti-Sirt2 (19655-1-AP) were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Immunolabeling, Cell Culture, Staining, Western Blot, Expressing, Control, Injection

Journal: Cell Communication and Signaling : CCS

Article Title: Kinesin KIFC3 is essential for microtubule stability and cytokinesis in oocyte meiosis

doi: 10.1186/s12964-024-01589-8

Figure Lengend Snippet: KIFC3 regulates spindle formation and chromosome alignment in mouse oocyte meiosis ( A ) Control and KIFC3-antibody injection oocytes at the MI stage were stained with anti-α-tubulin (green) and counterstained with Hoechst 33,342 to visualize the chromosomes (blue). Bar = 20 μm. ( B ) The rate of abnormal spindle morphology and misaligned chromosome after KIFC3-antibody injection at the MI stage. Graph shows means ± sem of results obtained in 3 independent experiments. * P < 0.05. ( C ) Thickness of the spindle middle plate. C indicates the maximal span of chromosomes; S indicates the maximal spindle length. Bar = 10 μm. The scattergram shows the C: S ratios for control and KIFC3-antibody injection oocytes at the MI stage. Graph shows means ± sem of results obtained in 3 independent experiments. *** P < 0.001. ( D ) Immunofluorescence analysis of γ-tubulin (red) and α-tubulin (green) in control and KIFC3-antibody injection oocytes. Bar = 10 μm. The histogram shows the percentages of control and KIFC3-antibody injection oocytes with abnormal γ-tubulin. Graph shows means ± sem of results obtained in 3 independent experiments. * P < 0.05. ( E ) Immunofluorescence analysis of p-Aurka (red) and α-tubulin (green) in control and KIFC3-antibody injection oocytes. Bar = 10 μm. The histogram shows the percentages of control and KIFC3-antibody injection oocytes with abnormal p-Aurka. Graph shows means ± sem of results obtained in 3 independent experiments. * P < 0.05

Article Snippet: The rabbit polyclonal anti-KIFC3 antibody (10125-2-AP) and rabbit polyclonal anti-Sirt2 (19655-1-AP) were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Control, Injection, Staining, Immunofluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Kinesin KIFC3 is essential for microtubule stability and cytokinesis in oocyte meiosis

doi: 10.1186/s12964-024-01589-8

Figure Lengend Snippet: KIFC3 regulates kinetochore-microtubule attachment in mouse oocytes ( A ) Control and KIFC3-antibody injection oocytes at the MI stage were stained with anti-α-tubulin (green), anti-CREST (red) and counterstained with Hoechst 33342 to visualize the chromosomes (blue). Bar = 10 μm. ( B ) The rate of abnormal kinetochore-microtubule attachment after KIFC3-antibody injection at the MI stage. Graph shows means ± sem of results obtained in 3 independent experiments. * P < 0.05. ( C ) Control and KIFC3-antibody injection oocytes at the MI stage were stained with anti-H3S10ph (red) and counterstained with Hoechst 33342 to visualize the chromosomes (blue). Bar = 5 μm. ( D ) The scattergram shows the relative fluorescence intensity of H3S10ph signals in control and KIFC3-antibody injection oocytes. Graph shows means ± sem of results obtained in 3 independent experiments. *** P < 0.001. ( E ) Control and KIFC3-antibody injection oocytes at the MI stage were stained with anti-BubR1 (green) and counterstained with Hoechst 33342 to visualize the chromosomes (blue). Bar = 5 μm. ( F ) The histogram shows the percentages of control and KIFC3-antibody injection oocytes with abnormal BubR1. Graph shows means ± sem of results obtained in 3 independent experiments. * P < 0.05. ( G ) Co-IP was performed with an anti-KIFC3 antibody. The immunoblots of protein precipitates were probed with an anti-Bub3 antibody. ( H ) Control and KIFC3-antibody injection oocytes at the MI stage were stained with anti-Bub3 (red) and counterstained with Hoechst 33342 to visualize the chromosomes (blue). Bar = 5 μm. ( I ) The histogram shows the percentages of control and KIFC3-antibody injection oocytes with abnormal Bub3. Graph shows means ± sem of results obtained in 3 independent experiments. ** P < 0.01

Article Snippet: The rabbit polyclonal anti-KIFC3 antibody (10125-2-AP) and rabbit polyclonal anti-Sirt2 (19655-1-AP) were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Control, Injection, Staining, Fluorescence, Co-Immunoprecipitation Assay, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Kinesin KIFC3 is essential for microtubule stability and cytokinesis in oocyte meiosis

doi: 10.1186/s12964-024-01589-8

Figure Lengend Snippet: KIFC3 regulates stability of spindle microtubules ( A ) Control and KIFC3-antibody injection oocytes after cold treatment at the MI stage were stained with anti-α-tubulin (green) and counterstained with Hoechst 33342 to visualize the chromosomes (blue). Bar = 20 μm. ( B ) The rate of abnormal spindle morphology after cold treatment of KIFC3 antibody injection and control oocytes at the MI stage. Graph shows means ± sem of results obtained in 3 independent experiments. * P < 0.05. ( C ) Control and KIFC3-antibody injection oocytes after Nocodazole treatment at the MI stage were stained with anti-α-tubulin (green) and counterstained with Hoechst 33342 to visualize the chromosomes (blue). Bar = 20 μm. ( D ) The relative area of spindle after nocodazole treatment of KIFC3 antibody injection and control oocytes at the MI stage. Graph shows means ± sem of results obtained in 3 independent experiments. * P < 0.05. ( E ) Co-IP was performed with an anti-KIFC3 antibody. The immunoblots of protein precipitates were probed with anti-Sitr2 and anti- acetylated tubulin antibody. ( F ) Immunofluorescence analysis of acetylated tubulin (red) and α-tubulin (green) in control and KIFC3 antibody injection oocytes. Bar = 10 μm. ( G ) The histogram shows the relative fluorescence intensity of acetylated tubulin signals in control and KIFC3 antibody injection oocytes. Graph shows means ± sem of results obtained in 3 independent experiments. ** P < 0.01. ( H ) Immunofluorescence analysis of Sirt2 (red) and α-tubulin (green) in control and KIFC3 antibody injection oocytes. Bar = 20 μm. ( I ) The histogram shows the relative fluorescence intensity of Sirt2 signals in control and KIFC3 antibody injection oocytes. Graph shows means ± sem of results obtained in 3 independent experiments. * P < 0.05

Article Snippet: The rabbit polyclonal anti-KIFC3 antibody (10125-2-AP) and rabbit polyclonal anti-Sirt2 (19655-1-AP) were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Control, Injection, Staining, Co-Immunoprecipitation Assay, Western Blot, Immunofluorescence, Fluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Kinesin KIFC3 is essential for microtubule stability and cytokinesis in oocyte meiosis

doi: 10.1186/s12964-024-01589-8

Figure Lengend Snippet: KIFC3 affects midbody formation and cytokinesis in mouse oocytes ( A ) Spindle morphology at the ATI stage and MII stage oocytes. Green, α-tubulin; blue, DNA. Repeat the experiment 4 times. Bar = 20 μm. ( B ) Co-IP was performed with an anti-KIFC3 antibody. The immunoblots of protein precipitates were probed with anti-PRC1 antibody. ( C ) Control and KIFC3-antibody injection oocytes at the ATI stage were stained with anti-α-tubulin (green), anti-PRC1 (red) and counterstained with Hoechst 33342 to visualize the chromosomes (blue). Repeat the experiment 3 times. Bar = 20 μm. ( D ) Diagram of the roles of KIFC3 during mouse oocyte meiotic maturation. KIFC3 regulated tubulin acetylation to stabilize the microtubules for spindle assembly and kinetochore-microtubules attachment, and KIFC3 also interacted with PRC1 for cytokinesis during mouse oocyte meiosis

Article Snippet: The rabbit polyclonal anti-KIFC3 antibody (10125-2-AP) and rabbit polyclonal anti-Sirt2 (19655-1-AP) were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Control, Injection, Staining

Journal: Biochimica et biophysica acta

Article Title: Identification of the kinesin KifC3 as a new player for positioning of peroxisomes and other organelles in mammalian cells.

doi: 10.1016/j.bbamcr.2013.08.002

Figure Lengend Snippet: Fig. 1. KifC3 and PEX1 interact in vivo. (A) PEX1-deficient human fibroblasts were transiently transfected with PEX1-myc and human KifC3, either with one plasmid alone or in combi- nation. 48 h after transfection cells were prepared for immunofluorescence and incubated with α-KifC3, α-tubulin α, α-myc, α-PEX14 antibodies and the corresponding fluorescence- tagged secondary antibodies. Overexpressed KifC3 was localized at microtubules (compare Fig. 2) whereas PEX1-myc was mainly found at peroxisomes. Upon coexpression of KifC3 and PEX1-myc, PEX1-myc was detected at microtubules where it colocalized with KifC3. (B) Interaction of KifC3 and PEX-myc was analyzed by coimmunoprecipitation (CoIp) using cell lysates of COS-7 cells transiently expressing PEX1-myc and KifC3, either alone or in combination. PEX1-myc was captured to magnetic beads via α-myc antibodies and was able to precipitate KifC3. Representative Western blots of CoIp input and supernatant samples (right panel) and eluates (left panel) using α-KifC3 and α-PEX1 antibodies are shown (repeated four times). Intensity of KifC3 eluate bands was quantified and revealed an intensity doubling when PEX1-myc and KifC3 were expressed in combination (100%) compared to samples expressing KifC3 alone (50%). Marked bands at 55 kDa (*) are caused by the applied IgGs and were used as loading controls for quantification. Scale bar: 10 μm.

Article Snippet: Primary antibodies were diluted as indicated and were applied to the cells for 30 min. After extensive washing cells were treated with secondary goat or donkey α-mouse or α-rabbit antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 594 (Molecular Probes, Invitrogen) for 10 min. Rabbit polyclonal antibodies against human KifC3 (ProteinTech Group) were used in a 1:50 dilution.

Techniques: In Vivo, Transfection, Plasmid Preparation, Incubation, Expressing, Magnetic Beads, Western Blot

Journal: Biochimica et biophysica acta

Article Title: Identification of the kinesin KifC3 as a new player for positioning of peroxisomes and other organelles in mammalian cells.

doi: 10.1016/j.bbamcr.2013.08.002

Figure Lengend Snippet: Fig. 2. Localization of endogenous and overexpressed KifC3. (A) COS-7 cells were processed for immunofluorescence microscopy using α-KifC3 antibodies. During interphase endogenous KifC3 localized near the nucleus and concentrated at perinuclear spots, which were most likely the microtubule organizing center (MTOC) as indicated by tubulin α staining. Visualization of peroxisomes by use of α-PMP70 antibodies did not reveal any colocalization of KifC3 with peroxisomes. During metaphase KifC3 was detected at the spindle fibers and spindle poles. (B) SDS-PAGE and western blot analysis using α-KifC3 antibodies revealed distinct expression levels of endogenous KifC3 in different cell lines with highest expression levels in A549 and COS-7 cells. To correct for the different loading of the total protein amount (HELA: 40 μg, A549: 10 μg, HEK 293 T: 40 μg, HepG2: 40 μg, GM5756T: 40 μg, COS-7: 13 μg, COS-7 + KifC3: 83 ng) additional analysis with α-MAPK (mitogen activated protein kinase) antibodies is shown. (C) Overexpression of KifC3 in COS-7 cells resulted in a microtubular staining pattern 24 h after transient transfection confirmed by colocalization with tubulin α. Just like endogenous KifC3, overexpressed KifC3 was not located at peroxisomes and it did not influence the peroxisomal distribution. Scale bar: 10 μm.

Article Snippet: Primary antibodies were diluted as indicated and were applied to the cells for 30 min. After extensive washing cells were treated with secondary goat or donkey α-mouse or α-rabbit antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 594 (Molecular Probes, Invitrogen) for 10 min. Rabbit polyclonal antibodies against human KifC3 (ProteinTech Group) were used in a 1:50 dilution.

Techniques: Microscopy, Staining, SDS Page, Western Blot, Expressing, Over Expression, Transfection

Journal: Biochimica et biophysica acta

Article Title: Identification of the kinesin KifC3 as a new player for positioning of peroxisomes and other organelles in mammalian cells.

doi: 10.1016/j.bbamcr.2013.08.002

Figure Lengend Snippet: Fig. 3. Clustering of peroxisomes after knockdown of KifC3. COS-7 cells were electroporated twice with siRNA targeting endogenous KifC3 or with non-target control siRNA (sictr), respec- tively. (A) Knockdown efficiencies on mRNA level were verified using quantitative real-time PCR measurements 24 h after the second electroporation (means and standard deviations are shown). KifC3-mRNA was reduced by 78 ± 0.08% (N = 14) whereas mRNA levels of Kif5B remained unaffected (N = 6). (B) Western blot analysis using α-KifC3 antibodies and α-MAPK antibodies as loading control revealed knockdown levels of KifC3 protein of 86 ± 12% (N = 11) a representative blot is shown. (C) 48 h after the second siRNA treatment cells were fixed and peroxisomes were stained using α-PEX14 antibodies. KifC3 knockdown samples showed increased amounts of cells with clustered peroxisomes compared to control cells whereas knockdown of Kif5B (knockdown Kif5B mRNA: 78.6 ± 3.34%, N = 2) even reduced the amount of cells with clustered peroxisomes. Means and standard deviations (KifC3: N = 4) or spreads (Kif5B: N = 2) are depicted. (D) Staining of peroxisomes and microtubules using α-PEX14 and α-tubulin α antibodies did not reveal any loss of microtubule-associated peroxisomes upon KifC3 knockdown compared to control cells. Scale bars: 10 μm.

Article Snippet: Primary antibodies were diluted as indicated and were applied to the cells for 30 min. After extensive washing cells were treated with secondary goat or donkey α-mouse or α-rabbit antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 594 (Molecular Probes, Invitrogen) for 10 min. Rabbit polyclonal antibodies against human KifC3 (ProteinTech Group) were used in a 1:50 dilution.

Techniques: Knockdown, Control, Real-time Polymerase Chain Reaction, Electroporation, Western Blot, Staining

Journal: Biochimica et biophysica acta

Article Title: Identification of the kinesin KifC3 as a new player for positioning of peroxisomes and other organelles in mammalian cells.

doi: 10.1016/j.bbamcr.2013.08.002

Figure Lengend Snippet: Fig. 4. Peroxisomal knockdown phenotype depends on microtubules. (A) Microtubules of COS-7 cells were depolymerized by incubation with nocodazole (noc) whereas addition of cytochalasin B (cyto B) degraded the actin skeleton. Controls were treated with DMSO, which was used as solvent. Microtubules were visualized using α-tubulin α antibodies and the actin skeleton was labeled by binding to rhodamine tagged phalloidin (ph–rh). (B) siRNA-treated COS-7 cells were incubated with cytochalasin B for 48 h, while nocodazole was added four hours prior to fixation. Controls were treated with DMSO for the respective times. Peroxisomes were stained using α-PEX14 antibodies. The observed increase of cells with perinuclear-clustered peroxisomes after KifC3 knockdown was independent of the actin skeleton, whereas microtubules were necessary. Cells with clustered peroxisomes of one experiment were quantified. At least 500 cells were counted. sictr: non-target control siRNA, scale bar: 10 μm.

Article Snippet: Primary antibodies were diluted as indicated and were applied to the cells for 30 min. After extensive washing cells were treated with secondary goat or donkey α-mouse or α-rabbit antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 594 (Molecular Probes, Invitrogen) for 10 min. Rabbit polyclonal antibodies against human KifC3 (ProteinTech Group) were used in a 1:50 dilution.

Techniques: Knockdown, Incubation, Solvent, Labeling, Binding Assay, Staining, Control

Journal: Biochimica et biophysica acta

Article Title: Identification of the kinesin KifC3 as a new player for positioning of peroxisomes and other organelles in mammalian cells.

doi: 10.1016/j.bbamcr.2013.08.002

Figure Lengend Snippet: Fig. 5. Cell cycle phase dependency of peroxisomal clustering. DNA of siRNA-treated A549 and COS-7 cells was stained using propidium iodide 48 h after the second electroporation. A histogram of fluorescence intensities was recorded by flow cytometry to quantify cells in a particular cell cycle phase corresponding to the amount of DNA. (A) Flow cytometry analysis of KifC3 knockdown COS-7 cells and control cells (sictr) did not show any differences concerning the distribution of the cells in the different cell cycle phases. (B) A549 cells were incubated with thymidine (thym) for 24 h to accumulate the cells in S-phase. The quantified relative amounts of cells in the particular cell cycle phases of thymidine-treated and untreated samples are depicted. The used thymidine block resulted in a shift of cell cycle phase distribution from G1- to S-phase and cells in S-phase were doubled. (C) SiRNA-treated A549 and COS-7 cells were incubated with thymidine (as in B) and the amount of cells with clustered peroxisomes was compared to unblocked cells. Means and standard deviations (COS −thym: N = 4) or spreads (COS + thym, A549 ± thym: N = 2) are depicted. At least 500 cells were counted.

Article Snippet: Primary antibodies were diluted as indicated and were applied to the cells for 30 min. After extensive washing cells were treated with secondary goat or donkey α-mouse or α-rabbit antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 594 (Molecular Probes, Invitrogen) for 10 min. Rabbit polyclonal antibodies against human KifC3 (ProteinTech Group) were used in a 1:50 dilution.

Techniques: Staining, Electroporation, Cytometry, Flow Cytometry, Knockdown, Control, Incubation, Blocking Assay

Journal: Biochimica et biophysica acta

Article Title: Identification of the kinesin KifC3 as a new player for positioning of peroxisomes and other organelles in mammalian cells.

doi: 10.1016/j.bbamcr.2013.08.002

Figure Lengend Snippet: Fig. 6. Altered morphology of mitochondria and endoplasmic reticulum after KifC3 knockdown. COS-7 cells were electroporated twice with KifC3-siRNA and with control siRNA (sictr), respectively and prepared for immunofluorescence microscopy 48 h after the second knockdown. Peroxisomes, early endosomes and the Golgi apparatus were stained using α-PEX14, α-EEA-1 and α-GM130 antibodies. To mark mitochondria cells were incubated with MitoTracker Red CMXRos for 30 min and the endoplasmic reticulum (ER) was visualized by transient transfection of the pDsRed2-ER vector 24 h prior fixation. Mitochondria and ER are artificially colored in green. Scale bar: 10 μm.

Article Snippet: Primary antibodies were diluted as indicated and were applied to the cells for 30 min. After extensive washing cells were treated with secondary goat or donkey α-mouse or α-rabbit antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 594 (Molecular Probes, Invitrogen) for 10 min. Rabbit polyclonal antibodies against human KifC3 (ProteinTech Group) were used in a 1:50 dilution.

Techniques: Knockdown, Control, Microscopy, Staining, Incubation, Transfection, Plasmid Preparation