Question 2 HER2 and HER3 belong to a family of receptor tyrosine kinases (RTKS). A representative cell signalling pathway involving HER2 and HER3 occurs as follows: (1) Ligand binds to HER3 resulting in its association with HER2. (2) HER2 phosphorylates HER3 at a tyrosine residue at position 1289 (Y1289). (3) Phosphorylated Y1289 (pY1289) recruits phosphoinositide 3-kinase (PI3-K). (4) The recruitment of PI3-K close to the plasma membrane leads to the recruitment of PDK and Akt to the plasma membrane. (5) PDK activates Akt, leading to various cellular outcomes, including proliferation and survival. HER2 NOTE Version 1 HER3 HER3 ligand (growth factor) HER20 HER3 HER3 ligand Lapatinib P13-K pY 1289 (2) - (3) P13-K a) HER2 expression is often elevated in cancer samples, and HER2 inhibitors have been developed as anti-cancer drugs. Lapatinib is one of them. You wish to examine whether inhibition of HER2 using Lapatinib also inhibits HER3 activation, following stimulation by the HER3 ligand. This can be achieved by observing levels of the phosphorylated form of HER3 in cells stimulated with HER3 ligand at the same time as inhibiting HER2 with Lapatinib. (4) Using numbered bullet-points, generate a protocol for Western blotting to address this question using reagents listed in Box 1 below. PDK Sample 2 + You need to prepare four types of cell extract, treated with HER3 ligand and Lapatinib as indicated in the table below: Sample 1 Sample 3 - Akt (5) Cell proliferation Cell survival + Sample 4 + + Page 3 of 5

Biochemistry

9th Edition

ISBN:9781319114671

Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Chapter1: Biochemistry: An Evolving Science

Section: Chapter Questions

Problem 1P

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Box one:

• Human breast cancer cell line BT474, which overexpresses HER2, in 10 cm Petri dishes
• Human HER3 ligand
• Lapatinib
• Lysis buffer A (containing 1% Triton and 0.1% SDS)
• Lysis buffer B (containing no detergent)
• Mouse anti-HER3 antibodies
• Mouse anti-pY1289 of HER3, that recognises the phospho-tyrosine at position
1289 of Her3
• Mouse anti-actin antibodies
• Horseradish peroxidase (HRP)-conjugated anti mouse IgG antibodies
• SDS-PAGE Sample buffer (contains SDS, mercaptoethanol and bromophenol
blue)
• Molecular weight markers
• SDS-PAGE gel
• Nitrocellulose membrane
• Phosphate buffered saline (PBS)
• Blocking buffer (5%w/v BSA in PBS)
• X-ray film
• Luminol/H2O2 (hydrogen peroxide