Anti rab9

Manufactured by Cell Signaling Technology

Anti-Rab9 is a primary antibody that specifically targets the Rab9 protein. Rab9 is a small GTPase that regulates the transport of cargo from late endosomes to the trans-Golgi network. The Anti-Rab9 antibody can be used to detect and study the localization and expression of Rab9 in various cellular and tissue samples.

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8 protocols using anti rab9

Protein Interaction Analysis Reagents

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Anti-FLAG and anti-Flag M1 agarose beads were from Sigma; anti-PPP2R1A and anti-Rab8, anti-Rab9 and Rab7 antibodies were from Cell Signaling. Anti-PPP2CA from Millipore, Inc. Peroxidase-conjugated anti-rabbit, anti-mouse and anti-goat secondary antibodies were from Jackson ImmunoResearch. Antibodies for immunofluorescence: anti-Rab8 and anti Rab9 were from Cell Signaling, anti-PPP2R1A was from Santa Cruz, anti-mouse Alexa Fluor-555-conjugated secondary antibody and anti-rabbit Alexa Fluor 488-conjugated secondary antibody were from Invitrogen. PPP2CA and PPP2R3C encoding plasmids were purchased from OpenBiosystem. PPP2R1A construct was kindly provided by Marc Vidal. RAB-GST cDNAs were kindly provided by Prof. Fukuda23 (link).

Sacco F., Mattioni A., Boldt K., Panni S., Santonico E., Castagnoli L., Ueffing M, & Cesareni G. (2016). A subset of RAB proteins modulates PP2A phosphatase activity. Scientific Reports, 6, 32857.

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Western Blot Analysis of Cellular Proteins

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Protein from the treated cell cultures was extracted and Western blot analysis was performed as described previously [37 (link)]. Expression of EBV lytic proteins was detected with anti-Zta (1:200; gift from Prof. P. Farrell, Imperial College, London, UK) and anti-BMRF1 (1:1000; gift from Dr.KH Chan, Department of Microbiology, HKU, Hong Kong SAR, China). Expression of phosphorylated ERK1/2 was detected with p-ERK1/2 rabbit polyclonal antibodies, (1:1000; #9101, Cell Signaling Technology Danvers, MA, USA). Expressions of ER stress markers PERK. IRE-1α, BiP, CHOP, XBP-1s was detected with ER stress sampler kit (1:1000; #9956, Cell Signaling Technology). Expression of autophagic proteins was detected with anti-ATG5 (1:1000; #12994, Cell Signaling Technology), anti-ATG7 (1:1000; #8558, Cell Signaling Technology,), anti-beclin-1 (1:1000; #3738, Cell Signaling Technology), anti-LC3B (1:1000; #2775, Cell Signaling Technology), anti-ATG10 (1:1000; #PA5-78593, Invitrogen), anti-ATG3 (1:1000; #3415, Cell Signaling Technology,), anti-ATG12 (1:1000; #4180, Cell Signaling Technology,) and anti-rab9 (1:1000; #5133. Cell Signaling Technology). Expression of human cellular α-tubulin and β-actin was detected with anti-α-tubulin and anti-β-actin antibody (1:5000; MilliporeSigma, Burlington, MA, USA), respectively, as loading controls.

Yiu S.P., Hui K.F., Münz C., Lo K.W., Tsao S.W., Kao R.Y., Yang D, & Chiang A.K. (2019). Autophagy-Dependent Reactivation of Epstein-Barr Virus Lytic Cycle and Combinatorial Effects of Autophagy-Dependent and Independent Lytic Inducers in Nasopharyngeal Carcinoma. Cancers, 11(12), 1871.

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Immunofluorescence staining of fixed cells

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Cells were fixed with 3.7% formaldehyde 20 mM HEPES in PBS for 10min. This step was skipped when working with Jurkat cells as they were fixed during the setup of the cells. The formaldehyde was quenched with 10 mM Tris in PBS for 10mins, and the cells were then permeabilised for 5mins with PBS containing 0.05% Tx-100. The cells were then blocked with 3% BSA in PBS for 20mins and then stained with primary antibodies (Ticilimumab, anti-LRBA (Atlas Antibodies), anti-Rab5, anti-Rab7, anti-Rab9 (all Cell Signaling Technology), anti-Rab11 (Invitrogen)) in 3mg/ml BSA in PBS for 1hr at RT. Next, goat anti-human IgG-AlexaFluor 546, donkey anti-rabbit IgG-AlexaFluor 488 (both Invitrogen), Hoechst 33342 (Thermo Fisher Scientific) and 2.5 μM CellTrace Violet (Molecular Probes) in 3mg/ml BSA in PBS were added to the cells for 45mins. All steps up to and including the 3% BSA block were carried out on ice, all the following steps were at RT, and the cells were washed 3 times with PBS between each step.

Janman D., Hinze C., Kennedy A., Halliday N., Waters E., Williams C., Rowshanravan B., Hou T.Z., Minogue S., Qureshi O.S, & Sansom D.M. (2021). Regulation of CTLA‐4 recycling by LRBA and Rab11. Immunology, 164(1), 106-119.

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Antibodies for Heparanase and Syndecan-1 Detection

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Rabbit polyclonal antibodies #1453 was prepared against purified latent 65 kDa heparanase [8 (link)]. Anti-heparanase monoclonal antibody was kindly provided by ImClone Systems (New York, NY). Anti-heparanase-2 antibody (20C5) has been described [15 (link)]. Rat anti-mouse syndecan-1 monoclonal antibody (clone 281-2) was kindly provided by Dr. Ralph D. Sanderson (UAB). This antibody is directed against the ectodomain of mouse syndecan-1 and is suitable for flow cytometry, immune staining and immunoblotting. Anti-vinculin and anti-actin monoclonal antibodies, phalloidin-TRITC, heparin, and methyl-β-cyclodextrin were purchased from Sigma (St. Louis, MO). Anti-α-actinin, anti-cortactin, anti-Rab7 and anti-Rab9 antibodies were from Cell Signaling (Beverly, MA). Anti-syntenin, anti-LAMP1, and anti-Myc Tag antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Latent 65 kDa heparanase was purified from medium conditioned by HEK-293 cells over expressing heparanase essentially as described [24 (link)].

Shteingauz A., Ilan N, & Vlodavsky I. (2014). Processing of heparanase is mediated by syndecan-1 cytoplasmic domain and involves syntenin and α-actinin. Cellular and molecular life sciences : CMLS, 71(22), 4457-4470.

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Immunofluorescence Analysis of Wing Disc

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Third instar wing discs were staged and fixed in 4% paraformaldehyde. Immuno-fluorescence was performed as described(Das et al., 2013 (link)). Antibodies used: anti-pRET[Y905], anti-pJnk, anti-pAkt, anti-SOX2, anti-slug, anti-NCadherin, anti-pEGFR[Y845], anti-pEGFR[Y1068], anti-pFGFR[Y653/654], anti-Rab5, anti-Rab7, anti-Rab9(Cell Signaling), anti-pSRC[Y418] (Invitrogen), anti-dpERK (SIGMA), plus anti-Actin,, anti-E-cadherin, anti-α-Catenin, anti-Rho1, anti-Syntaxin, anti-β-tubulin (Developmental Studies Hybridoma Bank), anti-Actin, anti-GAPDH antibodies (Santa Cruz Biotechnology), anti-Rac1 antibody (BD biosciences), anti-KIF5B(Abcam), anti-EGFR (Julia Cordero), anti-Arp3(William Theurkauf).

Das T.K, & Cagan R.L. (2017). KIF5B-RET Oncoprotein Signals Through A Multi-Kinase Signaling Hub. Cell reports, 20(10), 2368-2383.

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Multicolor Immunofluorescence Staining Protocol

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Cells were fixed with 3.7% formaldehyde 20 mM HEPES in PBS for 10 min. This step was skipped when working with Jurkat cells as they were fixed during the set‐up of the cells. The formaldehyde was quenched with 10 mM Tris in PBS for 10 min, and the cells were then permeabilized for 5 min with PBS containing 0.05% Triton‐X‐100. The cells were then blocked with 3% BSA in PBS for 20 min and then stained with primary antibodies (ticilimumab and anti‐LRBA (Atlas Antibodies); anti‐Rab5, anti‐Rab7 and anti‐Rab9 (all from Cell Signaling Technology); anti‐Rab11 (Invitrogen or Atlas Antibodies) and anti‐LC3A/B (Cell Signaling Technology)) in 3 mg/ml BSA in PBS for 1 h at RT. Next, goat anti‐human IgG‐Alexa Fluor 546, donkey anti‐rabbit IgG‐Alexa Fluor 488 (both Invitrogen), Hoechst 33342 (Thermo Fisher Scientific) and 2.5 µM CellTrace Violet (Molecular Probes) in 3 mg/ml BSA in PBS were added to the cells for 45 min. All steps up to and including the 3% BSA block were carried out on ice, all the following steps were at RT, and the cells were washed 3 times with PBS between each step.

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Integrin and Vesicle Trafficking Imaging Protocol

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The following primary antibodies were used for immunoblotting: anti-α5-integrin (Merck Millipore), anti-αv-integrin (Abcam, Cambridge, UK), anti-β1-integrin (BD Transduction Laboratories, San Jose, CA, USA), anti-β3-integrin (Merck Millipore), anti-β5-integrin (Abcam), anti-pERK (Cell Signaling Technology, Danvers, MA, USA), anti-ERK (Cell Signaling Technology), anti-LAMP1 (Cell Signaling Technology) and anti-β-actin (Sigma-Aldrich). The following primary antibodies were used for immunofluorescence staining: anti-α5-integrin (Cell Signaling Technology), anti-αv-integrin (Cell Signaling Technology), anti-LAMP1 (Cell Signaling Technology), anti-EEA1 (Cell Signaling Technology), anti-Rab5 (Cell Signaling Technology), anti-Rab7 (Cell Signaling Technology), anti-Rab9 (Cell Signaling Technology) and anti-γ-H2AX (Merck Millipore).

Wu P.H., Onodera Y., Ichikawa Y., Rankin E.B., Giaccia A.J., Watanabe Y., Qian W., Hashimoto T., Shirato H, & Nam J.M. (2017). Targeting integrins with RGD-conjugated gold nanoparticles in radiotherapy decreases the invasive activity of breast cancer cells. International Journal of Nanomedicine, 12, 5069-5085.

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Western Blotting of Protein Targets

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Proteins levels were analyzed by Western blotting using a standard protocol^{59 (link)}. Antibodies used in this study were: anti-Rab7 (9367S, 1:1000), anti-Alix (2171S, 1:1000), anti-CD81 (10037S, 56039S, 1:1000), anti-Tom20 (42406S, 1:1000), anti-Rab4 (2167S, 1:1000), anti-Rab5 (3547S, 1:1000), anti-Rab9 (5118S, 1:1000), anti-Rab11 (5589S, 1:1000), anti-LC3A/B (4108S, 1:1000), anti-Rab27 (69295S, 1:1000), anti-Arl8b (56085 S, 1:1000), anti-Atg5 (12994S, 1:1000), anti-Atg7 (2631S, 1:1000) from Cell Signaling Technology; anti-SQSTM1/p62 (ab56416, 1:1000), anti-Tsg101 (ab83, 1:1000), anti-Calreticulin (ab2907, 1:1000) from Abcam; anti-Tim23 (11123-1-AP, 1:1000) from Proteintech, anti-CD63 (PA5-92370, 1:500) from Invitrogen; anti-dendra2 (TA150090, 1:1000) from OriGene; anti-GAPDH (GTX627408, 1:2000) from GeneTex; Anti-Ubiquitin (P4D1) (sc-8017, 1:1000) from Santa Cruz, MTCO1 (459600, 1:1000) from Thermo Fisher Scientific, and MnSOD (06-984, 1:1000) from Millipore. Goat anti-mouse HRP (31430; 1:5000) and Goat-anti-rabbit HRP (31460, 1:5000) secondary antibodies from Thermo Fisher Scientific.

Liang W., Sagar S., Ravindran R., Najor R.H., Quiles J.M., Chi L., Diao R.Y., Woodall B.P., Leon L.J., Zumaya E., Duran J., Cauvi D.M., De Maio A., Adler E.D, & Gustafsson Å.B. (2023). Mitochondria are secreted in extracellular vesicles when lysosomal function is impaired. Nature Communications, 14, 5031.

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