Antibodies used were as follows: rabbit anti-human LC3 antibody (NB100-2220, Novus Biologicals, Madrid, Spain), mouse anti-human LAMP-1 antibody [H4A3] (ab 25630, Abcam, Cambridge, United Kingdom), mouse anti-human β-Glucocerebrosidase antibody (GBA) (B-6) (sc-166407, Santa Cruz, Heidelberg, Germany), rabbit recombinant anti-rab10 (phospho T73) antibody (ab230261, Abcam), rabbit monoclonal anti-Rab10 (8127S, Cell Signaling, Danvers, MA, United States), mouse anti-human β-Actin antibody (A1978-200UL, Sigma, Merck Life Science, Madrid, Spain), anti-rabbit antibody (A0545, Sigma, Merck Life Science, Madrid, Spain) and anti-mouse antibody (A3682, Sigma).
Ab230261
Manufactured by Abcam
Sourced in United States
Ab230261 is a lab equipment product offered by Abcam. It serves a core function, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
Nb100 2220, by Novus Biologicals (1 mentions)
A3682, by Merck Group (1 mentions)
Ab25630, by Abcam (1 mentions)
Ab51502, by Abcam (1 mentions)
Ab76442, by Abcam (1 mentions)
Ab19136, by Abcam (1 mentions)
Ab237703, by Abcam (1 mentions)
Ab241060, by Abcam (1 mentions)
Ab51252, by Abcam (1 mentions)
Mab1568, by Merck Group (1 mentions)
Sc 23948, by Santa Cruz Biotechnology (1 mentions)
Homer1, by Synaptic Systems (1 mentions)
Mab377, by Merck Group (1 mentions)
Rab10, by Cell Signaling Technology (1 mentions)
Ab5541, by Merck Group (1 mentions)
Vamp2, by Synaptic Systems (1 mentions)
Sc 58774, by Santa Cruz Biotechnology (1 mentions)
α synuclein, by BD (1 mentions)
Ab55080, by Abcam (1 mentions)
Ab108597, by Abcam (1 mentions)
6 protocols using ab230261
1
Antibody Characterization for Lysosomal Disorders
2
Comprehensive Antibody Panel for Neuronal Characterization
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Rab10 (4262S, Cell Signaling Technology) for immunoblot, Rab10 (ab237703, Abcam) for immunofluorescence, pRab10 for immunoblot (ab230261, Abcam), pRab10 for immunofluorescence (ab241060, Abcam), Hsc70 (ab19136, Abcam), α-Tubulin (SC23948, Santa Cruz Biotechnology), parvalbumin, (NBP2-50036, NovusBio), NeuN (MAB377, Millipore), SATB2 (ab51502 Abcam), calretinin (MAB1568, Millipore), DARPP32 (MAB4230, R&D Systems), ChAT (NBP2-46620, NovusBio), DAT loop (6-8D6 Santa Cruz Biotechnology), tyrosine hydroxylase (TH) (ab76442, Abcam), CD68 (NBP2-33337SS, NovusBio), GFAP (AB5541, Millipore), Olig2 (MABN50 Millipore), KDEL receptor (sc-58774, Santa Cruz Biotechnology), TGN46 (MA3-063, ThermoFisher), LAMP1 (1D4B, DHSB), EEA1 (NBP2-36568, NovusBio), α-Synuclein (610786, BD Transduction Lab), Synuclein (ab51252, Abcam), VAMP2 (104 211, Synaptic Systems), Homer1 (160 006 Synaptic Systems), Rab8a (ab188574).
3
Immunoblot Analysis of Lysosomal Markers
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Cells were lysed in cell lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5 mM EDTA, 0.5% (v/v) sodium deoxycholate, 1% (v/v) NP-40, pH 8) with protease and phosphatase inhibitor for 30 min. The lysates were centrifuged at 14,000 × g for 15 min at 4°C, and the supernatant protein was quantified using BCA assay. Total protein was normalized, mixed with 1x SDS-PAGE loading buffer, denatured at 95°C for 5 min and resolved on an SDS-polyacrylamide gel, and transferred to PVDF membrane. For dot blots, conditioned media was blotted on a nitrocellulose membrane without boiling. Membranes were blocked with 5% bovine serum albumin (Sigma). Blots were probed with primary antibodies to LAMP1 (abcam ab108597), GCase (abcam ab55080), pT73-Rab10 (abcam ab230261), Rab10 (cell signaling 8127S), pT72-Rab8a (abcam ab230260), Rab8a (abcam ab188574), pS935-LRRK2 (abcam 133450), LRRK2 (clone, 8629). αSyn oligomer (abcam ab209538). Secondary antibodies conjugated to horseradish peroxidase were used for detection using autoradiography.
4
PPM1H Regulation of Phospho-Rab10
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MEF cells stably expressing GFP-Rab10 were transiently transfected with plasmids carrying wild-type HA-PPM1H, HA-PPM1H[H153D], or HA-PPM1H[D288A]. After 24 h the cells were fixed with 4% (by vol) paraformaldehyde for 10 min, permeabilized with 0.1% saponin for 15 min, and blocked with 1% (by mass) BSA for 1 h. Cells were subsequently stained with mouse anti-HA antibody 1:1000 (Sigma-Aldrich H3663) and rabbit phospho-Rab10 1:1000 (Abcam ab230261). Highly cross absorbed H+L secondary antibodies (Life Technologies) conjugated to Alexa 568 or Alexa 647 were used at 1:5000. Primary and secondary antibody incubations were for 1 h at room temperature. All images were obtained using a spinning disk confocal microscope (Yokogawa) with an electron multiplying charge coupled device (EMCCD) camera (Andor, UK) and a 20x1.4NA or 40x1.4NA objective. Images were analyzed using CellProfiler and presented as maximum intensity projections. Results were quantified by determining the ratios of phospho-Rab10 signal to GFP-Rab10 in cells expressing wild-type HA-PPM1H, HA-PPM1H[H153D], or HA-PPM1H[D288A], and normalizing these numbers to the phospho-Rab10/GFP-Rab10 ratio in non-expressing cells. For each condition at least 20 cells were analyzed. Significance was determined by one-way analysis of variance with Dunnett's post-test at 95% confidence interval. ***, P < 0.001.
5
Immunofluorescence and Western Blotting of LRRK2, Rab8, and Rab10
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The following primary antibodies were used: rabbit anti-LRRK2 (Abcam, Cambridge, UK; ab133474; 1:100 dilution for immunofluorescence (IF) and 1:1000 for western blotting (WB)), mouse anti-Rab8 (BD; 610844; 1:200 for IF and 1:2000 for WB), rabbit anti-Rab8 phospho-T72 (Abcam; ab230260; 1:500 for WB), mouse anti-LAMP1 (DSBH, Iowa City, IA, USA; H4A3; 1:50 for IF), rabbit anti-Rab10 (Abcam; ab237703; 1:100 for IF and 1:1000 for WB), rabbit anti-Rab10 phospho-T73 (Abcam; ab230261; 1:100 for IF and 1:1000 for WB), anti-FLAG tag (Sigma-Aldrich; F1804; 1:1000 for WB), anti-Myc tag (Cell Signaling Technology, Danvers, MA, USA; 2276; 1:2000 for WB), anti-β-actin (Sigma-Aldrich; A5441; 1:5000 for WB), rabbit anti-p62 (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan; PM045, 1:1000 for WB), and rabbit anti-LC3B (Cell Signaling; 3868, 1:2000 for WB).
6
Protein Extraction and Analysis Protocol
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The protein in TE-1 and KYSE30 cells was extracted using RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific) and the concentration was assessed by BCA Protein Assay Kit (Beyotime, Shanghai, China). Subsequently, the protein samples were separated by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membrane (GE Healthcare, Piscataway, NJ, USA). The membrane was blocked in no-fat milk for 4 hrs and then incubated with primary antibody overnight at 4°C. The membrane was incubated with secondary antibody at 37°C for another 2 hrs. RapidStep ECL Reagent (Millipore) was used to detect the fluorescence intensity. Rab10 (1:1000, ab230261), β-actin (1:1000, ab8227) and secondary antibody (1:25,000; ab97051) antibodies were obtained from Abcam (Cambridge, MA, USA).
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