The following antibodies were used: antibodies against Drp1 (D6C7) (#8570S; WB 1:1000, IF 1:100, IP/PLA 1:100), P-Drp1(S616) (#3455S; WB 1:1000), P-Drp1 (S637) (#4867S; WB 1:1000), Desmin (D93F5) (5332S; WB 1:1000, IF 1:100), eIF2alpha (#9722S; WB 1:1000) and P-eIF2alpha (S51) (#9721; WB 1:1000), Cdc2 (#28439; WB 1:1000) and P-Cdc2 (Thr161) (#9114; WB 1:1000) which were purchased from Cell Signaling Technology (Beverly, MA, USA); antibodies against KLC1 (L2) (sc-58776; WB 1:500 IF 1:100) and GAPDH (FL-335) (sc-25778; WB 1:3000) which were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); antibodies against alpha-Tubulin (ab11304-100; IF/PLA 1:100), KIF5B (ab167429; WB 1:1000, IF/PLA 1:200, IP 1:100), and KLC1 (ab174273; IF/PLA 1:100) which were purchased from Abcam (Cambridge, UK); an antibody against DLP1 (i.e. Drp1) (611113: WB 1:800) which was purchased from BD Biosciences; antibodies against Desmin (D1033; WB 1:500) and Clpp (WH0008192M1; WB: 1:1000) which were purchased from Sigma-Aldrich; an antibody against OxPhos Complex IV subunit IV (i.e. COX IV) (20E8C12; WB 1:2000, IF 1:50) which was purchased from Invitrogen (Carlsbad, CA); the antibody against p-desmin Ser-31 (#D375-3; WB 1:1000, IF 1:500) was purchased from MBL Life Science (Woburn, MA, USA).
Ab167429
Manufactured by Abcam
Sourced in United States
Ab167429 is a laboratory equipment product manufactured by Abcam. It is designed to perform a specific function in a laboratory setting. The core function of this product is to [CORE FUNCTION].
Automatically generated - may contain errors
Lab products found in correlation
Gapdh fl 335 sc 25778, by Santa Cruz Biotechnology (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Penicillin, by Sartorius (1 mentions)
Ab205606, by Abcam (1 mentions)
L glutamine, by Sartorius (1 mentions)
Jetpei, by Polyplus Transfection (1 mentions)
Supersignal west pico plus chemiluminescence substrate, by Thermo Fisher Scientific (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Mitotracker orange cmtmros, by Thermo Fisher Scientific (1 mentions)
F actin, by Thermo Fisher Scientific (1 mentions)
Rhodamine phalloidin dye, by Thermo Fisher Scientific (1 mentions)
α tubulin, by Abcam (1 mentions)
Ab52866, by Abcam (1 mentions)
α actinin, by Santa Cruz Biotechnology (1 mentions)
Ab190638, by Abcam (1 mentions)
Endoa2, by Santa Cruz Biotechnology (1 mentions)
His tag, by Cell Signaling Technology (1 mentions)
Kif3a, by Santa Cruz Biotechnology (1 mentions)
Protein a g beads, by GE Healthcare (1 mentions)
Nitrocellulose nc membrane, by Merck Group (1 mentions)
5 protocols using ab167429
1
Antibody Panel for Mitochondrial Dynamics
2
AAV-mediated Syne4 protein analysis
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HEK293 cells were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% FBS, 1% penicillin, and 1% L-glutamine (Biological Industries), transfected with AAV. Syne4WD, AAV. Syne4AA, or empty FLAG control plasmids, using jetPEI (Polyplus) according to manufacturer’s instructions and harvested 48 h after transfection. One 10 cm plate per condition was lysed 1 ml RIPA buffer (Sigma) and Halt Protease Inhibitor Cocktail (Thermo). Tubes were placed on an end-over-end shaker for 1 h at 4°C followed by centrifugation at 16,000 g for 15 min at 4°C. 20 µl of the resulting supernatant was used as input and the rest was incubated with EZview anti-FLAG M2 beads (F2426, Sigma) for 1-2 h at 4°C on an end-over-end shaker. Beads were then washed and eluted in sample buffer according to manufacturer’s instructions and loaded into a 10% SDS-PAGE gel. Samples were then transferred onto a nitrocellulose membrane which was then blocked in 3% skimmed milk (BD Difco). Membranes were then subjected to immunoblotting. Kif5b was detected by anti Kif5b (Abcam, ab167429). FLAG was detected using rabbit anti DDDDK antibody (Abcam, ab205606). Blots were visualized using anti rabbit HRP antibody (Cell Signaling Technologies, 7074) and SuperSignal West Pico PLUS Chemiluminescence Substrate (Thermo Scientific).
3
Immunofluorescence Staining Protocol
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For immunofluorescence staining, the cells were fixed with 4% paraformaldehyde for 15 min in PBS, permeabilised with 0.2% Triton X-100 in PBS, blocked with 5% BSA in PBS and then stained with primary antibodies, α-tubulin (ab52866, Abcam, Cambridge, MA, USA), α-actinin (A7811, Santa Cruz Biotech, CA, USA) or KIF5B (ab167429, Abcam), followed by Alexa Fluor 546/633 secondary antibodies. Rhodamine phalloidin dye (R415, Invitrogen) and WGA Alexa Fluor® 488 conjugate (WGA, W11261, Invitrogen) were incubated with the cells for 20 min at room temperature for F-actin or cell membrane staining. To detect the mitochondrial transfer shown in Fig. 5a , MitoTracker® Orange CMTMRos (Mito Orange, M7510 Invitrogen) was incubated with the cells for 20 min at 37 °C.
Stained cells were visualised and analysed using a laser scanning confocal microscope with a 63X/1.4NA oil immersion objective lens and excitation wavelengths of 488, 549 and 633 nm. All images were acquired using ZEN 2012 software.
Stained cells were visualised and analysed using a laser scanning confocal microscope with a 63X/1.4NA oil immersion objective lens and excitation wavelengths of 488, 549 and 633 nm. All images were acquired using ZEN 2012 software.
4
Co-Immunoprecipitation Assay for Protein-Protein Interactions
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The Co-IP test was evaluated in accordance with previously provided instructions [24 (link)]. Transfected HEK293 cells or DRG tissues were lysed in cold Co-IP RIPA buffer. The lysates were centrifuged, and 5% of each supernatant was taken for the input sample. The remaining supernatants were incubated with 5–10 μg EndoA2 (mouse, Santa Cruz, USA, sc-365704), Piezo2 (rabbit, 1:200, Alomone, Israel, APC-090) or His (mouse, Santa Cruz, USA, sc-8036) antibody at 4 °C overnight and then with protein A/G beads (GE Healthcare, UK) at 4 °C for 4 h. The immunoprecipitated samples were denatured and prepared for immunoblotting. Immunoprecipitation was performed with antibodies against Piezo2 (rabbit, 1:1000, Novus, USA, NBP1-78,624; rabbit, 1:200, Alomone, Israel, APC-090), TACAN/Tmem120a (rabbit, 1:1000, Bioss, China, bs-19952R), ASIC2 (rabbit, 1:1000, Bioss, China, bs-4915R), ASIC3 (rabbit, 1:1000, Abcam, USA, ab190638), KIF5B (rabbit, 1:2000, Abcam, USA, ab167429), KIF5A (rabbit, 1:1000, Abcam, USA, ab5628), KIF17 (rabbit, 1:1000, Bioss, China, bs-3527R), KIF3A (goat, 1:100, Santa Cruz, USA, sc-18745), KIF3B (rabbit, 1:1000, Bioss, China, bs-17085R), His-tag (rabbit, 1:1000, Cell Signaling Technology, USA, 12,698) or myc-tag (rabbit, 1:2000, Bioss, China, bs-23166R). The precipitant was washed, denatured and prepared for immunoblotting.
5
Western Blot Analysis of Colon Proteins
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Total protein was extracted from human colon tissues using a Sangon kit (Sangon Biotech, Shanghai, China). Equal amounts of denatured protein were separated with 10% SDS-PAGE (Sangon Biotech) and then transferred to a nitrocellulose (NC) membrane (Millipore Corporation). The NC membranes were blocked with 10% skimmed milk for 1 h at RT and incubated with primary antibodies (anti-ARF4, 1:1000 dilution, ab171746, Abcam; anti-KIF5B, 1:1000 dilution, ab167429, Abcam; anti-RAB8A, 1:3000 dilution, ab188574, Abcam; and anti-β-actin, 1:1000 dilution, #3700, CST) at 4 °C overnight. After rinsing, the NC membranes were further incubated with the appropriate secondary antibody for 2 h at RT. An ECL reagent (GE Healthcare) was applied to visualize the band signals. The grayscale values of the bands were normalized to the values of the corresponding β-actin band to determine the expression levels of the proteins.
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