Past exam of the biochemistry and Molecular Biology course of the University of Cambridge / 2025 / Paper 2 / Section B / 13

Culture each patient and control sample under matched conditions, including equivalent growth-factor withdrawal where appropriate, then prepare paired unstimulated and PDGF-stimulated samples with the same stimulation time. Rapidly lyse on ice with suitable detergents and protease and phosphatase inhibitors. Measure protein concentration and load equal amounts after SDS-PAGE sample preparation. Separate the proteins with molecular-mass markers, transfer them to a suitable membrane, verify transfer and block non-specific binding. Incubate with the Ras-PST25 primary antibody, wash, add a detectable secondary antibody, wash again and record the signal within its linear range. Preserve the original phosphorylation state and compare equal, verifiably transferred inputs. Include a total-protein loading measure, appropriate antibody controls and independent biological replicates; use matched exposures rather than independently rescaling lanes.

The healthy sample establishes the basal and normal stimulated patterns for the same cell type and handling conditions. Sample A contains bands around $96$ and $30\mathrm{kDa}$ in both conditions; PDGF additionally induces bands near $73$, $62$ and $44$–$45\mathrm{kDa}$. These are compatible, respectively, with stimulated Raf, Akt and unresolved MEK/ERK signals, given the supplied masses. Ligand-dependent appearance of those bands is the normal comparison for suspected constitutive signaling. Their identities remain provisional until confirmed with target-specific reagents. The unchanged bands are useful internal observations but cannot be assumed to be validated loading controls merely because their intensities look similar.

The most informative difference is phosphorylation without PDGF, relative to sample A. The following are candidate interpretations of the supplied Western blot, not definitive clinical diagnoses or protein identifications:

A single broad phospho-antibody cannot reliably distinguish the closely sized MEK and ERK bands or determine whether any phosphorylation site is activating. Clinical assessment and genetic and functional confirmation remain necessary.

Use a total-protein stain or validated constitutive loading control, and measure total amounts of the candidate signaling proteins alongside their site-specific phosphorylated forms. This distinguishes increased phosphorylation fraction from increased protein abundance. Include a no-primary or secondary-only control and a phosphatase-treated aliquot to test phospho-dependent detection. Confirm candidate identities using validated target-specific phospho-antibodies, suitable positive controls and, where feasible, depletion or inhibition of the relevant target. Repeat the paired time course with biological replicates and non-saturated imaging. Matching culture state, stimulus dose and sampling time is essential because an ordinary difference in response kinetics could otherwise mimic constitutive activation.

An activating Ras lesion is a parsimonious candidate, because Ras lies upstream of the branch to Raf and the branch to phosphoinositide 3-kinase. Persistent GTP-bound Ras can therefore produce both basal patterns through a single change. A lesion restricted to MEK would not directly explain the simultaneous Akt signal. The blot does not uniquely locate the mutation: a constitutively active upstream receptor tyrosine kinase or altered regulation of Ras could also activate both branches. Test the Ras nucleotide state, sequence relevant genes, and determine which targeted perturbation suppresses both outputs.

mTOR has the supplied molecular mass of about $259\mathrm{kDa}$, so optimize the separation and transfer for a much larger protein. Use a suitably low-percentage or gradient SDS-PAGE gel and high-mass markers, and verify efficient transfer with a protocol appropriate for large proteins, such as optimized wet transfer. Probe with a validated phospho-mTOR reagent and measure total mTOR in parallel. The broad Ras-PST25 reagent cannot be assumed to recognize a useful site on mTOR merely because it recognizes other pathway proteins. Include a positive pathway control and a suitable inhibition or phosphatase control; a missing band on the existing blot could reflect poor separation or transfer rather than absent mTOR phosphorylation.

Analyze the corresponding region from a parallel preparative SDS-PAGE gel, digest the recovered proteins and identify peptides by liquid chromatography-mass spectrometry. The lane and gel region should match the $35\mathrm{kDa}$ signal, with an adjacent-background or control-sample region processed in parallel. Because several proteins can comigrate, validate the candidate against the actual phospho-band, for example by immunoprecipitating the candidate and testing Ras-PST25 reactivity, using a target-specific Western blot, or showing disappearance of the band after specific depletion. Phosphopeptide identification provides additional evidence; mass alone or detecting a common contaminant in the excised region is insufficient.

PDGFR is a receptor tyrosine kinase; its key ligand-induced autophosphorylation is on tyrosine residues. Ras-PST25 recognizes phosphorylated serine residues and threonine residues, so it is not the appropriate reagent to detect the characteristic activated PDGFR phosphotyrosines. Use a phosphotyrosine or validated phospho-PDGFR antibody, ideally alongside total PDGFR. The larger receptor may also require a different gel and transfer range, but changing the size range alone would not correct the epitope mismatch.