Western Blot:Article Title: Centrin-POC5 inner scaffold provides distal centriole integrity for sperm flagellar assembly
Article Snippet: The cells were cultured in Dulbecco’s modified Eagle’s medium (Nacalai Tesque, 08459-64) supplemented with 10% FBS (Biowest, S1780-500, lot: S15064S1780), 1 mM sodium pyruvate (Nacalai Tesque, 06977-34), penicillin (100 U/ml), and streptomycin (100 μg/ml; Nacalai Tesque, 09367-34) at 37°C in a 5% CO 2 atmosphere. .. The following primary antibodies were used: rabbit polyclonal antibodies against POC5 [this study; 1:500 for IF, 1:1000 for immunoblotting (IB), and 1:250 for U-ExM], SFI1 (Proteintech, 13550-1-AP; 1:250 for U-ExM), POC1A (this study; 1:250 for U-ExM), POC1B (this study; 1:250 for U-ExM), centrin1 (this study; for IP), centrin2 (this study; for IP), CP110 (Proteintech, 12780-1-AP; 1:250 for U-ExM), CEP164 (Novus Biologicals, NBP1-81445, lot: 000012571; 1:500 for IF), CEP164 (Proteintech, 22227-1-AP, lot: 00045133; 1:250 for U-ExM), α-tubulin (MBL, PM054, lot: 011; 1:500 for IF), pericentrin (Abcam, ab4448; 1:1000 for IF), and SYCP1 (Abcam, ab15090, lot: GR3272415-4; 1:1000 for IF); chicken polyclonal antibody against SYCP3 [Hiroki Shibuya Lab ( ); 1:3000 for IF and 1:1000 for U-ExM]; sheep polyclonal antibody against C2CD3 (R&D Systems, AF7348, lot: CFQX0122121; 1:125 for U-ExM); mouse monoclonal antibodies against centrin (Merck Millipore, 04-1624, clone: 20H5; 1:1000 for IF, 1:5000 for IB, and 1:500 for U-ExM), β-actin (Sigma-Aldrich, A2228-200UL, clone: AC-74; 1:2000 for IB), γ-tubulin (Abcam, ab27074, clone: TU-30; 1:500 for IF), Polyglutamylation modification (AdipoGen, AG-20B-0020-C100, clone: GT335; 1:1000 for IF), and acetylated tubulin (Sigma-Aldrich, T6793-100UL, clone: 6-11B-1; 1:1000 for IF); guinea pig monoclonal antibodies against α-tubulin (ABCD Antibodies, AA345; 1:250 for U-ExM) and β-tubulin (ABCD Antibodies, AA344; 1:250 for U-ExM); and rat monoclonal antibody against centrin2 [BioLegend, 698602, clone: W16110A; 1:200 for IF]. .. The following secondary antibodies were used: Alexa Fluor 488 donkey anti-mouse immunoglobulin G (IgG; H + L) (Thermo Fisher Scientific, A21202; 1:500 for IF), Alexa Fluor 488 donkey anti-rabbit IgG (H + L) (Thermo Fisher Scientific, A21206; 1:500 for IF), Alexa Fluor 488 goat anti-guinea pig IgG (H + L) (Thermo Fisher Scientific, A11073; 1:800 for U-ExM), Alexa Fluor 488 donkey anti-sheep IgG (H + L) (Thermo Fisher Scientific, A11015; 1:800 for U-ExM), Alexa Fluor 488 goat anti-chicken IgG (H + L) (Thermo Fisher Scientific, A32931; 1:500 for IF), Alexa Fluor 488 donkey anti-rat IgG (H + L) (Thermo Fisher Scientific, A21208; 1:500 for IF), Alexa Fluor 555 donkey anti-mouse IgG (H + L) (Thermo Fisher Scientific, A31570 ; 1:500 for IF and 1:800 for U-ExM), Alexa Fluor 555 donkey anti-rabbit IgG (H + L) (Thermo Fisher Scientific, A31572; 1:500 for IF and 1:800 for U-ExM), Alexa Fluor 555 goat anti-guinea pig IgG (H + L) (Thermo Fisher Scientific, A21435; 1:800 for U-ExM), Alexa Fluor 647 donkey anti-mouse IgG (H + L) (Thermo Fisher Scientific, A31571; 1:800 for U-ExM), Alexa Fluor 647 donkey anti-rabbit IgG (H + L) (Thermo Fisher Scientific, A31573 ; 1:800 for U-ExM), Alexa Fluor 647 donkey anti-chicken IgG (H + L) (Thermo Fisher Scientific, A78952; 1:500 for IF and 1:800 for U-ExM), horseradish peroxidase (HRP)–conjugated goat polyclonal antibodies against mouse IgG (Sigma-Aldrich, A4416-1ML; 1:5000 for IB), and HRP-conjugated goat polyclonal antibodies against rabbit IgG (Sigma-Aldrich, A0545-1ML; 1:5000 for IB).
Article Title: Centrin-POC5 inner scaffold provides distal centriole integrity for sperm flagellar assembly.
Article Snippet: The cells were cultured in Dulbecco’s modified Eagle’s medium (Nacalai Tesque, 08459- 64) supplemented with 10% FBS (Biowest, S1780- 500, lot: S15064S1780), 1 mM sodium pyruvate (Nacalai Tesque, 06977- 34), penicillin (100 U/ ml), and streptomycin (100 μg/ml; Nacalai Tesque, 09367- 34) at 37°C in a 5% CO2 atmosphere. .. The following primary antibodies were used: rabbit polyclonal antibodies against POC5 [this study; 1:500 for IF, 1:1000 for immunoblotting (IB), and 1:250 for U- ExM], SFI1 (Proteintech, 13550- 1- AP; 1:250 for U- ExM), POC1A (this study; 1:250 for U- ExM), POC1B (this study; 1:250 for U- ExM), centrin1 (this study; for IP), centrin2 (this study; for IP), CP110 (Proteintech, 12780- 1- AP; 1:250 for UExM), CEP164 (Novus Biologicals, NBP1- 81445, lot: 000012571; 1:500 for IF), CEP164 (Proteintech, 22227- 1- AP, lot: 00045133; 1:250 for U- ExM), α- tubulin (MBL, PM054, lot: 011; 1:500 for IF), pericentrin (Abcam, ab4448; 1:1000 for IF), and SYCP1 (Abcam, ab15090, lot: GR3272415- 4; 1:1000 for IF); chicken polyclonal antibody against SYCP3 [Hiroki Shibuya Lab (56); 1:3000 for IF and 1:1000 for U- ExM]; sheep polyclonal antibody against C2CD3 (R&D Systems, AF7348, lot: CFQX0122121; 1:125 for U- ExM); mouse monoclonal antibodies against centrin (Merck Millipore, 04- 1624, clone: 20H5; 1:1000 for IF, 1:5000 for IB, and 1:500 for U- ExM), β- actin (SigmaAldrich, A2228- 200UL, clone: AC- 74; 1:2000 for IB), γ- tubulin (Abcam, ab27074, clone: TU- 30; 1:500 for IF), Polyglutamylation modification (AdipoGen, AG- 20B- 0020- C100, clone: GT335; 1:1000 for IF), and acetylated tubulin (Sigma- Aldrich, T6793- 100UL, clone: 6- 11B- 1; 1:1000 for IF); guinea pig monoclonal antibodies against α- tubulin (ABCD Antibodies, AA345; 1:250 for U- ExM) and βtubulin (ABCD Antibodies, AA344; 1:250 for U- ExM); and rat monoclonal antibody against centrin2 [BioLegend, 698602, clone: W16110A; 1:200 for IF]. .. The following secondary antibodies were used: Alexa Fluor 488 donkey anti- mouse immunoglobulin G (IgG; H + L) (Thermo Fisher Scientific, A21202; 1:500 for IF), Alexa Fluor 488 donkey anti- rabbit IgG (H + L) (Thermo Fisher Scientific, A21206; 1:500 for IF), Alexa Fluor 488 goat anti- guinea pig IgG (H + L) (Thermo Fisher Scientific, A11073; 1:800 for U- ExM), Alexa Fluor 488 donkey anti- sheep IgG (H + L) (Thermo Fisher Scientific, A11015; 1:800 for U- ExM), Alexa Fluor 488 goat anti- chicken IgG (H + L) (Thermo Fisher Scientific, A32931; 1:500 for IF), Alexa Fluor 488 donkey anti- rat IgG (H + L) (Thermo Fisher Scientific, A21208; 1:500 for IF), Alexa Fluor 555 donkey anti- mouse IgG (H + L) (Thermo Fisher Scientific, A31570; 1:500 for IF and 1:800 for U- ExM), Alexa Fluor 555 donkey anti- rabbit IgG (H + L) (Thermo Fisher Scientific, A31572; 1:500 for IF and 1:800 for U- ExM), Alexa Fluor 555 goat anti- guinea pig IgG (H + L) (Thermo Fisher Scientific, A21435; 1:800 for U- ExM), Alexa Fluor 647 donkey anti- mouse IgG (H + L) (Thermo Fisher Scientific, A31571; 1:800 for U- ExM), Alexa Fluor 647 donkey anti- rabbit IgG (H + L) (Thermo Fisher Scientific, A31573; 1:800 for U- ExM), Alexa Fluor 647 donkey anti- chicken IgG (H + L) (Thermo Fisher Scientific, A78952; 1:500 for IF and 1:800 for U- ExM), horseradish peroxidase (HRP)–conjugated goat polyclonal antibodies against mouse IgG (Sigma- Aldrich, A44161ML; 1:5000 for IB), and HRP- conjugated goat polyclonal antibodies against rabbit IgG (Sigma- Aldrich, A0545- 1ML; 1:5000 for IB).
Bioprocessing:Article Title: Centrin-POC5 inner scaffold provides distal centriole integrity for sperm flagellar assembly
Article Snippet: The cells were cultured in Dulbecco’s modified Eagle’s medium (Nacalai Tesque, 08459-64) supplemented with 10% FBS (Biowest, S1780-500, lot: S15064S1780), 1 mM sodium pyruvate (Nacalai Tesque, 06977-34), penicillin (100 U/ml), and streptomycin (100 μg/ml; Nacalai Tesque, 09367-34) at 37°C in a 5% CO 2 atmosphere. .. The following primary antibodies were used: rabbit polyclonal antibodies against POC5 [this study; 1:500 for IF, 1:1000 for immunoblotting (IB), and 1:250 for U-ExM], SFI1 (Proteintech, 13550-1-AP; 1:250 for U-ExM), POC1A (this study; 1:250 for U-ExM), POC1B (this study; 1:250 for U-ExM), centrin1 (this study; for IP), centrin2 (this study; for IP), CP110 (Proteintech, 12780-1-AP; 1:250 for U-ExM), CEP164 (Novus Biologicals, NBP1-81445, lot: 000012571; 1:500 for IF), CEP164 (Proteintech, 22227-1-AP, lot: 00045133; 1:250 for U-ExM), α-tubulin (MBL, PM054, lot: 011; 1:500 for IF), pericentrin (Abcam, ab4448; 1:1000 for IF), and SYCP1 (Abcam, ab15090, lot: GR3272415-4; 1:1000 for IF); chicken polyclonal antibody against SYCP3 [Hiroki Shibuya Lab ( ); 1:3000 for IF and 1:1000 for U-ExM]; sheep polyclonal antibody against C2CD3 (R&D Systems, AF7348, lot: CFQX0122121; 1:125 for U-ExM); mouse monoclonal antibodies against centrin (Merck Millipore, 04-1624, clone: 20H5; 1:1000 for IF, 1:5000 for IB, and 1:500 for U-ExM), β-actin (Sigma-Aldrich, A2228-200UL, clone: AC-74; 1:2000 for IB), γ-tubulin (Abcam, ab27074, clone: TU-30; 1:500 for IF), Polyglutamylation modification (AdipoGen, AG-20B-0020-C100, clone: GT335; 1:1000 for IF), and acetylated tubulin (Sigma-Aldrich, T6793-100UL, clone: 6-11B-1; 1:1000 for IF); guinea pig monoclonal antibodies against α-tubulin (ABCD Antibodies, AA345; 1:250 for U-ExM) and β-tubulin (ABCD Antibodies, AA344; 1:250 for U-ExM); and rat monoclonal antibody against centrin2 [BioLegend, 698602, clone: W16110A; 1:200 for IF]. .. The following secondary antibodies were used: Alexa Fluor 488 donkey anti-mouse immunoglobulin G (IgG; H + L) (Thermo Fisher Scientific, A21202; 1:500 for IF), Alexa Fluor 488 donkey anti-rabbit IgG (H + L) (Thermo Fisher Scientific, A21206; 1:500 for IF), Alexa Fluor 488 goat anti-guinea pig IgG (H + L) (Thermo Fisher Scientific, A11073; 1:800 for U-ExM), Alexa Fluor 488 donkey anti-sheep IgG (H + L) (Thermo Fisher Scientific, A11015; 1:800 for U-ExM), Alexa Fluor 488 goat anti-chicken IgG (H + L) (Thermo Fisher Scientific, A32931; 1:500 for IF), Alexa Fluor 488 donkey anti-rat IgG (H + L) (Thermo Fisher Scientific, A21208; 1:500 for IF), Alexa Fluor 555 donkey anti-mouse IgG (H + L) (Thermo Fisher Scientific, A31570 ; 1:500 for IF and 1:800 for U-ExM), Alexa Fluor 555 donkey anti-rabbit IgG (H + L) (Thermo Fisher Scientific, A31572; 1:500 for IF and 1:800 for U-ExM), Alexa Fluor 555 goat anti-guinea pig IgG (H + L) (Thermo Fisher Scientific, A21435; 1:800 for U-ExM), Alexa Fluor 647 donkey anti-mouse IgG (H + L) (Thermo Fisher Scientific, A31571; 1:800 for U-ExM), Alexa Fluor 647 donkey anti-rabbit IgG (H + L) (Thermo Fisher Scientific, A31573 ; 1:800 for U-ExM), Alexa Fluor 647 donkey anti-chicken IgG (H + L) (Thermo Fisher Scientific, A78952; 1:500 for IF and 1:800 for U-ExM), horseradish peroxidase (HRP)–conjugated goat polyclonal antibodies against mouse IgG (Sigma-Aldrich, A4416-1ML; 1:5000 for IB), and HRP-conjugated goat polyclonal antibodies against rabbit IgG (Sigma-Aldrich, A0545-1ML; 1:5000 for IB).
Article Title: Centrin-POC5 inner scaffold provides distal centriole integrity for sperm flagellar assembly.
Article Snippet: The cells were cultured in Dulbecco’s modified Eagle’s medium (Nacalai Tesque, 08459- 64) supplemented with 10% FBS (Biowest, S1780- 500, lot: S15064S1780), 1 mM sodium pyruvate (Nacalai Tesque, 06977- 34), penicillin (100 U/ ml), and streptomycin (100 μg/ml; Nacalai Tesque, 09367- 34) at 37°C in a 5% CO2 atmosphere. .. The following primary antibodies were used: rabbit polyclonal antibodies against POC5 [this study; 1:500 for IF, 1:1000 for immunoblotting (IB), and 1:250 for U- ExM], SFI1 (Proteintech, 13550- 1- AP; 1:250 for U- ExM), POC1A (this study; 1:250 for U- ExM), POC1B (this study; 1:250 for U- ExM), centrin1 (this study; for IP), centrin2 (this study; for IP), CP110 (Proteintech, 12780- 1- AP; 1:250 for UExM), CEP164 (Novus Biologicals, NBP1- 81445, lot: 000012571; 1:500 for IF), CEP164 (Proteintech, 22227- 1- AP, lot: 00045133; 1:250 for U- ExM), α- tubulin (MBL, PM054, lot: 011; 1:500 for IF), pericentrin (Abcam, ab4448; 1:1000 for IF), and SYCP1 (Abcam, ab15090, lot: GR3272415- 4; 1:1000 for IF); chicken polyclonal antibody against SYCP3 [Hiroki Shibuya Lab (56); 1:3000 for IF and 1:1000 for U- ExM]; sheep polyclonal antibody against C2CD3 (R&D Systems, AF7348, lot: CFQX0122121; 1:125 for U- ExM); mouse monoclonal antibodies against centrin (Merck Millipore, 04- 1624, clone: 20H5; 1:1000 for IF, 1:5000 for IB, and 1:500 for U- ExM), β- actin (SigmaAldrich, A2228- 200UL, clone: AC- 74; 1:2000 for IB), γ- tubulin (Abcam, ab27074, clone: TU- 30; 1:500 for IF), Polyglutamylation modification (AdipoGen, AG- 20B- 0020- C100, clone: GT335; 1:1000 for IF), and acetylated tubulin (Sigma- Aldrich, T6793- 100UL, clone: 6- 11B- 1; 1:1000 for IF); guinea pig monoclonal antibodies against α- tubulin (ABCD Antibodies, AA345; 1:250 for U- ExM) and βtubulin (ABCD Antibodies, AA344; 1:250 for U- ExM); and rat monoclonal antibody against centrin2 [BioLegend, 698602, clone: W16110A; 1:200 for IF]. .. The following secondary antibodies were used: Alexa Fluor 488 donkey anti- mouse immunoglobulin G (IgG; H + L) (Thermo Fisher Scientific, A21202; 1:500 for IF), Alexa Fluor 488 donkey anti- rabbit IgG (H + L) (Thermo Fisher Scientific, A21206; 1:500 for IF), Alexa Fluor 488 goat anti- guinea pig IgG (H + L) (Thermo Fisher Scientific, A11073; 1:800 for U- ExM), Alexa Fluor 488 donkey anti- sheep IgG (H + L) (Thermo Fisher Scientific, A11015; 1:800 for U- ExM), Alexa Fluor 488 goat anti- chicken IgG (H + L) (Thermo Fisher Scientific, A32931; 1:500 for IF), Alexa Fluor 488 donkey anti- rat IgG (H + L) (Thermo Fisher Scientific, A21208; 1:500 for IF), Alexa Fluor 555 donkey anti- mouse IgG (H + L) (Thermo Fisher Scientific, A31570; 1:500 for IF and 1:800 for U- ExM), Alexa Fluor 555 donkey anti- rabbit IgG (H + L) (Thermo Fisher Scientific, A31572; 1:500 for IF and 1:800 for U- ExM), Alexa Fluor 555 goat anti- guinea pig IgG (H + L) (Thermo Fisher Scientific, A21435; 1:800 for U- ExM), Alexa Fluor 647 donkey anti- mouse IgG (H + L) (Thermo Fisher Scientific, A31571; 1:800 for U- ExM), Alexa Fluor 647 donkey anti- rabbit IgG (H + L) (Thermo Fisher Scientific, A31573; 1:800 for U- ExM), Alexa Fluor 647 donkey anti- chicken IgG (H + L) (Thermo Fisher Scientific, A78952; 1:500 for IF and 1:800 for U- ExM), horseradish peroxidase (HRP)–conjugated goat polyclonal antibodies against mouse IgG (Sigma- Aldrich, A44161ML; 1:5000 for IB), and HRP- conjugated goat polyclonal antibodies against rabbit IgG (Sigma- Aldrich, A0545- 1ML; 1:5000 for IB).
Modification:Article Title: Centrin-POC5 inner scaffold provides distal centriole integrity for sperm flagellar assembly
Article Snippet: The cells were cultured in Dulbecco’s modified Eagle’s medium (Nacalai Tesque, 08459-64) supplemented with 10% FBS (Biowest, S1780-500, lot: S15064S1780), 1 mM sodium pyruvate (Nacalai Tesque, 06977-34), penicillin (100 U/ml), and streptomycin (100 μg/ml; Nacalai Tesque, 09367-34) at 37°C in a 5% CO 2 atmosphere. .. The following primary antibodies were used: rabbit polyclonal antibodies against POC5 [this study; 1:500 for IF, 1:1000 for immunoblotting (IB), and 1:250 for U-ExM], SFI1 (Proteintech, 13550-1-AP; 1:250 for U-ExM), POC1A (this study; 1:250 for U-ExM), POC1B (this study; 1:250 for U-ExM), centrin1 (this study; for IP), centrin2 (this study; for IP), CP110 (Proteintech, 12780-1-AP; 1:250 for U-ExM), CEP164 (Novus Biologicals, NBP1-81445, lot: 000012571; 1:500 for IF), CEP164 (Proteintech, 22227-1-AP, lot: 00045133; 1:250 for U-ExM), α-tubulin (MBL, PM054, lot: 011; 1:500 for IF), pericentrin (Abcam, ab4448; 1:1000 for IF), and SYCP1 (Abcam, ab15090, lot: GR3272415-4; 1:1000 for IF); chicken polyclonal antibody against SYCP3 [Hiroki Shibuya Lab ( ); 1:3000 for IF and 1:1000 for U-ExM]; sheep polyclonal antibody against C2CD3 (R&D Systems, AF7348, lot: CFQX0122121; 1:125 for U-ExM); mouse monoclonal antibodies against centrin (Merck Millipore, 04-1624, clone: 20H5; 1:1000 for IF, 1:5000 for IB, and 1:500 for U-ExM), β-actin (Sigma-Aldrich, A2228-200UL, clone: AC-74; 1:2000 for IB), γ-tubulin (Abcam, ab27074, clone: TU-30; 1:500 for IF), Polyglutamylation modification (AdipoGen, AG-20B-0020-C100, clone: GT335; 1:1000 for IF), and acetylated tubulin (Sigma-Aldrich, T6793-100UL, clone: 6-11B-1; 1:1000 for IF); guinea pig monoclonal antibodies against α-tubulin (ABCD Antibodies, AA345; 1:250 for U-ExM) and β-tubulin (ABCD Antibodies, AA344; 1:250 for U-ExM); and rat monoclonal antibody against centrin2 [BioLegend, 698602, clone: W16110A; 1:200 for IF]. .. The following secondary antibodies were used: Alexa Fluor 488 donkey anti-mouse immunoglobulin G (IgG; H + L) (Thermo Fisher Scientific, A21202; 1:500 for IF), Alexa Fluor 488 donkey anti-rabbit IgG (H + L) (Thermo Fisher Scientific, A21206; 1:500 for IF), Alexa Fluor 488 goat anti-guinea pig IgG (H + L) (Thermo Fisher Scientific, A11073; 1:800 for U-ExM), Alexa Fluor 488 donkey anti-sheep IgG (H + L) (Thermo Fisher Scientific, A11015; 1:800 for U-ExM), Alexa Fluor 488 goat anti-chicken IgG (H + L) (Thermo Fisher Scientific, A32931; 1:500 for IF), Alexa Fluor 488 donkey anti-rat IgG (H + L) (Thermo Fisher Scientific, A21208; 1:500 for IF), Alexa Fluor 555 donkey anti-mouse IgG (H + L) (Thermo Fisher Scientific, A31570 ; 1:500 for IF and 1:800 for U-ExM), Alexa Fluor 555 donkey anti-rabbit IgG (H + L) (Thermo Fisher Scientific, A31572; 1:500 for IF and 1:800 for U-ExM), Alexa Fluor 555 goat anti-guinea pig IgG (H + L) (Thermo Fisher Scientific, A21435; 1:800 for U-ExM), Alexa Fluor 647 donkey anti-mouse IgG (H + L) (Thermo Fisher Scientific, A31571; 1:800 for U-ExM), Alexa Fluor 647 donkey anti-rabbit IgG (H + L) (Thermo Fisher Scientific, A31573 ; 1:800 for U-ExM), Alexa Fluor 647 donkey anti-chicken IgG (H + L) (Thermo Fisher Scientific, A78952; 1:500 for IF and 1:800 for U-ExM), horseradish peroxidase (HRP)–conjugated goat polyclonal antibodies against mouse IgG (Sigma-Aldrich, A4416-1ML; 1:5000 for IB), and HRP-conjugated goat polyclonal antibodies against rabbit IgG (Sigma-Aldrich, A0545-1ML; 1:5000 for IB).
Article Title: Centrin-POC5 inner scaffold provides distal centriole integrity for sperm flagellar assembly.
Article Snippet: The cells were cultured in Dulbecco’s modified Eagle’s medium (Nacalai Tesque, 08459- 64) supplemented with 10% FBS (Biowest, S1780- 500, lot: S15064S1780), 1 mM sodium pyruvate (Nacalai Tesque, 06977- 34), penicillin (100 U/ ml), and streptomycin (100 μg/ml; Nacalai Tesque, 09367- 34) at 37°C in a 5% CO2 atmosphere. .. The following primary antibodies were used: rabbit polyclonal antibodies against POC5 [this study; 1:500 for IF, 1:1000 for immunoblotting (IB), and 1:250 for U- ExM], SFI1 (Proteintech, 13550- 1- AP; 1:250 for U- ExM), POC1A (this study; 1:250 for U- ExM), POC1B (this study; 1:250 for U- ExM), centrin1 (this study; for IP), centrin2 (this study; for IP), CP110 (Proteintech, 12780- 1- AP; 1:250 for UExM), CEP164 (Novus Biologicals, NBP1- 81445, lot: 000012571; 1:500 for IF), CEP164 (Proteintech, 22227- 1- AP, lot: 00045133; 1:250 for U- ExM), α- tubulin (MBL, PM054, lot: 011; 1:500 for IF), pericentrin (Abcam, ab4448; 1:1000 for IF), and SYCP1 (Abcam, ab15090, lot: GR3272415- 4; 1:1000 for IF); chicken polyclonal antibody against SYCP3 [Hiroki Shibuya Lab (56); 1:3000 for IF and 1:1000 for U- ExM]; sheep polyclonal antibody against C2CD3 (R&D Systems, AF7348, lot: CFQX0122121; 1:125 for U- ExM); mouse monoclonal antibodies against centrin (Merck Millipore, 04- 1624, clone: 20H5; 1:1000 for IF, 1:5000 for IB, and 1:500 for U- ExM), β- actin (SigmaAldrich, A2228- 200UL, clone: AC- 74; 1:2000 for IB), γ- tubulin (Abcam, ab27074, clone: TU- 30; 1:500 for IF), Polyglutamylation modification (AdipoGen, AG- 20B- 0020- C100, clone: GT335; 1:1000 for IF), and acetylated tubulin (Sigma- Aldrich, T6793- 100UL, clone: 6- 11B- 1; 1:1000 for IF); guinea pig monoclonal antibodies against α- tubulin (ABCD Antibodies, AA345; 1:250 for U- ExM) and βtubulin (ABCD Antibodies, AA344; 1:250 for U- ExM); and rat monoclonal antibody against centrin2 [BioLegend, 698602, clone: W16110A; 1:200 for IF]. .. The following secondary antibodies were used: Alexa Fluor 488 donkey anti- mouse immunoglobulin G (IgG; H + L) (Thermo Fisher Scientific, A21202; 1:500 for IF), Alexa Fluor 488 donkey anti- rabbit IgG (H + L) (Thermo Fisher Scientific, A21206; 1:500 for IF), Alexa Fluor 488 goat anti- guinea pig IgG (H + L) (Thermo Fisher Scientific, A11073; 1:800 for U- ExM), Alexa Fluor 488 donkey anti- sheep IgG (H + L) (Thermo Fisher Scientific, A11015; 1:800 for U- ExM), Alexa Fluor 488 goat anti- chicken IgG (H + L) (Thermo Fisher Scientific, A32931; 1:500 for IF), Alexa Fluor 488 donkey anti- rat IgG (H + L) (Thermo Fisher Scientific, A21208; 1:500 for IF), Alexa Fluor 555 donkey anti- mouse IgG (H + L) (Thermo Fisher Scientific, A31570; 1:500 for IF and 1:800 for U- ExM), Alexa Fluor 555 donkey anti- rabbit IgG (H + L) (Thermo Fisher Scientific, A31572; 1:500 for IF and 1:800 for U- ExM), Alexa Fluor 555 goat anti- guinea pig IgG (H + L) (Thermo Fisher Scientific, A21435; 1:800 for U- ExM), Alexa Fluor 647 donkey anti- mouse IgG (H + L) (Thermo Fisher Scientific, A31571; 1:800 for U- ExM), Alexa Fluor 647 donkey anti- rabbit IgG (H + L) (Thermo Fisher Scientific, A31573; 1:800 for U- ExM), Alexa Fluor 647 donkey anti- chicken IgG (H + L) (Thermo Fisher Scientific, A78952; 1:500 for IF and 1:800 for U- ExM), horseradish peroxidase (HRP)–conjugated goat polyclonal antibodies against mouse IgG (Sigma- Aldrich, A44161ML; 1:5000 for IB), and HRP- conjugated goat polyclonal antibodies against rabbit IgG (Sigma- Aldrich, A0545- 1ML; 1:5000 for IB).
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