Sv5 pk1

Protein extracts for Western blotting were prepared following cell fixation with trichloroacetic acid and bead beating. Extracts were then separated by SDS–polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes. Antibodies used for Western detection were α-Clb5 (Santa Cruz Biotechnology, sc20170), α-Clb2 (Santa Cruz Biotechnology, sc9071), α-Sic1 (Santa Cruz Biotechnology, sc50441), α-Orc6 (clone SB49), α-Orc2 [a gift from S. P. Bell (71 (link))], α-Rga2 and α-Boi1 [a gift from D. McCusker (51 (link))], α-Cdc24 [a gift from M. Peter (72 (link))], α-myc (clone 9E10), α–hemagglutinin (HA) (clone 12CA5), α-Pk (Bio-Rad, clone SV5-Pk1; Abcam, ab15828), and α-tubulin (Crick cell services, clone TAT-1).

Proteins separated by SDS-PAGE, were transferred onto PVDF membranes and immunoblotted with antibodies anti-double phosphorylated ERK1/2 (dilution 1:5000) (#9101, Cell Signalling Technologies, Danvers, MA, USA) and anti-phosphoTyr ERK (dilution 1:1000) (#sc-7383, Santa Cruz Biotechnology Inc., Dallas, TX, USA). After cell transfection, anti-V5 tag (dilution 1:1000) (#ab 27671, SV5-Pk1, Abcam, Cambridge, UK) was used to detect specifically recombinant over-expressed ERK2 protein. Anti-complex III or TOM40 (dilution 1:5000) (#ab 14745, Abcam, Cambridge, UK) and anti-polimerase II (POLII) or anti-TFIID (or TBP) (#ab sc-421, Santa Cruz Biotechnology Inc., Dallas, TX, USA) (dilution 1:1000) were used as mitochondrial and nuclear loading control respectively, and antibodies against actin (dilution 1:4000) were used for cytosolic fraction characterization (Upstate, Fisher Scientific, Thermo Fischer Scientific, #05661). Cells were then incubated with secondary horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies (dilution 1:5000; #1706515 and #1721011, respectively; Bio-Rad Laboratories Inc.). Chemiluminescence was developed with enhanced ECL reagent (GE Healthcare, Buckinghamshire, UK) and bands were detected by autoradiography (X100 Autoradiography film, GE Healthcare). Acrylamide and PVDF membranes were from Bio-Rad Laboratories Inc.

Anti‐α‐tubulin (T9026 Sigma‐Aldrich Co.), anti‐neuron‐specific class III β‐tubulin (Tuj1, TUBB3 #80120, BioLegend) anti‐acetylated tubulin (T7451 Sigma‐Aldrich), anti‐GAPDH (MAB374 Merck Millipore) anti‐PKR (anti‐EIF2AK2 Antibody No. ABIN500507, antibodies‐online.com), anti‐V5 tag antibody (SV5‐Pk1, Abcam), anti‐tau antibody (in house produced polyclonal Tau antibody, affinity‐purified rabbit towards human 2N4R tau), anti‐tau (A0024, DAKO), anti‐STAT1 (#9172 Cell Signaling Technology), anti‐pSTAT1 (pS727, #9177 Cell Signaling Technology), anti‐pS199/202 tau (44‐768G Thermo Fisher Scientific), anti‐pS396 tau (44‐752G Thermo Fisher Scientific), AT8 (pS202/T205 tau, MN1020 Thermo Fisher Scientific), AT180 (pT231 tau, MN1040 Thermo Fisher Scientific), AT270 (pT181 tau, MN1050 Thermo Fisher Scientific), anti‐pS262 (44‐750G Thermo Fisher Scientific), anti‐pS404 (44‐758G Thermo Fisher Scientific), anti‐pS409 (Lu0041G, kindly provided by Lundbeck) and anti‐pS422 (ab79415, Abcam) were used in accordance with the manufacturers' recommendations. HRP‐conjugated rabbit immunoglobulins (#P0217, Dako) and secondary HRP conjugated mouse immunoglobulins (#P0260, Dako) were used as secondary antibodies.