In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Summary

Small ubiquitin-related modifier (SUMO) family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. This paper describes a protocol for the detection of retinoblastoma (Rb) protein SUMOylation under endogenous and exogenous conditions in human cells.

Abstract

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, small ubiquitin-related modifier (SUMO) is a ubiquitin-like protein that is covalently attached to the lysine residues of a variety of target proteins to regulate cellular processes, such as gene transcription, DNA repair, protein interaction and degradation, subcellular transport, and signal transduction. The most common approach to detecting protein SUMOylation is based on the expression and purification of recombinant tagged proteins in bacteria, allowing for an in vitro biochemical reaction which is simple and suitable for addressing mechanistic questions. However, due to the complexity of the process of SUMOylation in vivo, it is more challenging to detect and analyze protein SUMOylation in cells, especially when under endogenous conditions. A recent study by the authors of this paper revealed that endogenous retinoblastoma (Rb) protein, a tumor suppressor that is vital to the negative regulation of the cell cycle progression, is specifically SUMOylated at the early G1 phase. This paper describes a protocol for the detection and analysis of Rb SUMOylation under both endogenous and exogenous conditions in human cells. This protocol is appropriate for the phenotypical and functional investigation of the SUMO-modification of Rb, as well as many other SUMO-targeted proteins, in human cells.

Introduction

The accurate control of cell cycle progression in eukaryotic cells is based on a tight regulatory network, which ensures that particular events take place in an ordered manner^1,2. One of the key players in this network is the retinoblastoma (Rb) protein, the first cloned tumor suppressor^1,3. The Rb protein is thought to be a negative regulator of cell cycle progression, especially for the G0/G1 to S phase transition, and tumor growth^4,5. Failure of Rb function either directly leads to the most ....

Protocol

1. Detection of Endogenous Rb SUMOylation at the Early G1 Phase

1. Cell culture and cell cycle synchronization.
   1. Maintain HEK293 cells in growth medium containing Dulbecco's Modified Eagle Medium (DMEM) supplemented with 1% Pen-Strep and 10% fetal bovine serum (FBS) at 37 °C and 5% CO₂ in an incubator.
   2. Synchronize the HEK293 cells at the G0 phase.
      1. Count the HEK293 cells using a hemocytometer and seed ~1.5 x 10⁷ cells in a 15 cm dish with 25ml growth medium for 24 h before treatment. After reaching a confluence of 70% - 80%, aspirate the medium and wash the cells twice wit....

Results

To detect endogenous Rb SUMOylation during cell cycle progression, this study first synchronized HEK293 cells at five different stages of the cell cycle (G0, early G1, G1, S, and G2/M) as described in the protocol section of this paper. The quality of synchronization was confirmed by using the nucleic acid stain with propidium iodide followed by flow cytometry analysis (Figure 1). Next, the cells were collected and lysed by denaturing RIPA buffer.The SUMO pro.......

Discussion

This paper describes a protocol to detect and analyze the endogenous SUMOylation of Rb in human cells. As this method is specifically focused on the endogenous Rb protein without any alternation of global SUMO-related signal, it is an important tool for investigating Rb-SUMO modification under completely natural physiologic circumstances.

To achieve this aim, it is important to keep in mind that: 1) although SUMO comprises four isoforms (SUMO1-4, each encoded by different genes)in comparison t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's Modified Eagle Medium (DMEM)	Thermo Fisher Scientific	11995065
Opti-MEM	Thermo Fisher Scientific	31985070
Fetal Bovine Serum (FBS)	Thermo Fisher Scientific	26140079
Penicillin-Streptomycin	Thermo Fisher Scientific	15140122
Phosphate-buffered Saline (PBS)	Thermo Fisher Scientific	10010023
Trypsin-EDTA	Thermo Fisher Scientific	25200056
Thymidine	Sigma	T9250
Nocodazole	Sigma	M1404
propidium iodide	Thermo Fisher Scientific	P3566
Triton X-100	AMRESCO	694
RNase A	Thermo Fisher Scientific	EN0531
N-Ethylmaleimide	Sigma	E3876
Sodium Dodecyl Sulfate (SDS)	AMRESCO	M107
Nonidet P-40 Substitute (NP-40)	AMRESCO	M158
protease inhibitor	Roche	5892970001
Mouse Immunoglobulin G (IgG)	Santa Cruz Biotechnology	sc-2025
Rb antibody	Cell Signaling Technology	#9309
Protein A/G-Sepharose Beads	Santa Cruz Biotechnology	sc-2003
Lipofectamine-2000	Thermo Fisher Scientific	11668019
Nickel Nitrilotriacetic Acid (Ni-NTA) Agarose Beads	Qiagen	30230
Imidazole	Sigma	I0250
4%-20% Gradient SDS-PAGE Gel	BIO-RAD	4561096
Polyvinylidene Difluoride (PVDF) Membrane	Millipore	IPVH00010
Tween-20	AMRESCO	M147
Tubulin antibody	Abmart	M30109
SUMO1 antibody	Thermo Fisher Scientific	33-2044
GFP antibody	Abmart	M20004
Horseradish Peroxidase (HRP) secondary antibody	Jackson ImmunoResearch Laboratories	715-035-150
enhanced chemiluminescence (ECL) Kit	Thermo Fisher Scientific	32106