白细胞内整合素依赖的RhoB激活实验

Janne Lehtonen; Katarzyna Potrzebowska; Malin Samuelsson; Lena Svensson

doi:10.21769/BioProtoc.3105

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Peer-reviewed

Integrin Dependent RhoB Activation Assay Using Leukocytes

白细胞内整合素依赖的RhoB激活实验

JL Janne Lehtonen

KP Katarzyna Potrzebowska

MS Malin Samuelsson

LS Lena Svensson email

发布: 2018年12月05日第8卷第23期 DOI: 10.21769/BioProtoc.3105 浏览次数: 2877

评审: Vaibhav B. ShahWoojong LeeAnonymous reviewer(s)

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Abstract

To be able to migrate, leukocyte needs to re-use its adhesion molecules to move forward. These adhesion molecules are called integrins and are intracellularly transported via endocytosis and exocytosis in order to translocate to a new site on the cell membrane. The intracellular transportation is regulated by different small GTPases including RhoB. Here we describe an activation assay of RhoB in leukocytes migrating on ICAM-1Fc coated dishes using commercially available Rhoteikin coated agarose beads. Although this is a specific protocol for LFA-1 induced RhoB activation, it can also be used for RhoB activation induced by other soluble and insoluble factors.

Keywords: Leukocytes (白细胞)

LFA-1 (LFA-1)

RhoB (RhoB)

Small GTPases (小G蛋白)

Rhoteikin (Rhoteikin)

Pull-down (下拉)

Background

Leukocytes are frequently migrating to clear infections and/or to become activated in the lymph nodes. For a cell to move forward, it needs to be able to attach and reuse its integrins. These steps involve various small GTPases that control the intracellular transport of proteins. Small GTPases switch from active to inactive form by hydrolysis of GTP into GDP, which is controlled by specific GTPase-activating proteins (GAPs) and Guanine nucleotide exchange factors (GEFs) (Cavalli et al., 2001). RhoB is one of these important small GTPases that regulates endosomal trafficking of receptors in different cell types including leukocytes (Fernandez-Borja et al., 2005). We have recently found that the major integrin used by T lymphocytes, Lymphocyte function-associated antigen 1 (LFA-1) is recycled by a Rab11-dependent pathway that is controlled by RhoB activity (Samuelsson et al., 2017). Here, we present a protocol to measure RhoB activation based on the pulldown of active RhoB with commercially available Rhotekin-coated agarose beads. The Rhotekin-RBD protein specifically recognizes and binds to the active, GTP-bound form of the Rho protein. Furthermore, we describe integrin-dependent RhoB activation using coverslips coated with the integrin ligand ICAM-1Fc.

Materials and Reagents

Pipette tips
Coverslips (32 mm diameter) (VWR, catalog number: 631-0162)
6-well dish (SARSTEDT, catalog number: 833920)
Microfuge tubes (SARSTEDT, catalog number: 72690.301)
96-well plate (SARSTEDT, catalog number: 821581.0019)
PVDF membrane (Thermo Fisher Scientific, catalog number: 88518)
Leukocytes (here we use human primary T lymphoblasts purified from whole blood)
RhoB Activation Assay Kit (Cellbiolabs, catalog number: STA-403-B)
ICAM-1Fc (Biolegend, catalog number: 552904)
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A1933)
Pierce^TM Coomassie Plus Bradford assay reagent and BSA-standard (Thermo Fisher Scientific, catalog numbers: 23238 and 23209, respectively)
Primary antibodies
1. RhoB (Santa Cruz Biotechnology, catalog number: sc-180) or use the RhoB antibody included in the RhoB Activation Assay Kit
2. LFA-1 (m38) (GeneTex, catalog number: GTX26132)
3. α-Actin (Sigma-Aldrich, catalog number: A5441)
Secondary antibodies
1. Goat anti-Rabbit IgG-HRP (Thermo Fisher Scientific, catalog number: 31460)
2. Sheep anti-Mouse IgG-HRP (GE Healthcare, Amersham, catalog number: NA9310V)
Pierce^TMECLplus Western Blotting substrate (Thermo Fisher Scientific, catalog number: 32132)
70% ethanol
5% milk powder
Methanol
Buffers
1. HEPES (Thermo Fisher Scientific, catalog number: 15630080)
2. Protease inhibitors (cOmplete^TMMini EDTA-free Protease Inhibitor Cocktail, Roche) (Sigma-Aldrich, catalog number: 11836170001)
3. Tween 20 (Thermo Fisher Scientific, catalog number: TA-125-TW)
4. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, catalog number: 14040133)
5. Hank’s balanced salt solution (HBSS) with or without Ca²⁺/Mg²⁺(Thermo Fisher Scientific, Gibco, catalog numbers: 14025092 and 14175095)
6. 2x reducing SDS-PAGE buffer (Sigma-Aldrich, catalog number: S3401-10VL)
7. HBSS wash buffer (see Recipes)
8. Migration buffer (see Recipes)

Equipment

Pipettes
Forceps
Centrifuges (refrigerated microcentrifuge and swing out centrifuge with plate holders)
Rocker/shaker
Plate reader capable of measuring absorbance at the wavelength appropriate to the protein assay (e.g., 595 nm for Protein Assay)
Electrophoresis system
Immunoblotting system
Incubator

Software

ImageJ (NIH)

Procedure

English

中文翻译

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如何引用

Lehtonen, J., Potrzebowska, K., Samuelsson, M. and Svensson, L. (2018). Integrin Dependent RhoB Activation Assay Using Leukocytes. Bio-protocol 8(23): e3105. DOI: 10.21769/BioProtoc.3105.