Erk1 2

Cells were lysed in 1x RIPA buffer (9806 Cell Signaling) containing 10 mM PMSF, Protease Inhibitor Cocktail (M250 Amresco), Phosphatase Inhibitor Cocktail 2 (P5726 Milipore), and Phosphatase Inhibitor Cocktail 3 (P0044 Milipore) followed by immunoblotting using LI-COR Odyssey® CLx Imaging System. Antibodies were typically duplexed using rabbit antibodies for phosphorylated antibodies and mouse antibodies for total protein. Li-Cor secondary antibodies, Goat anti-Rabbit IRDye 680RD and Goat anti-Mouse IRDye 800CW, were used with the duplexed primary antibodies.
All primary antibodies were rabbit unless specified and were sourced as follows: PTPRS (mouse Cat.No.ab55640 Abcam); PTPRS (goat AF3430 R&D Systems); alpha-Tubulin (mouse sc-8035 Santa Cruz). All other antibodies were obtained from Cell Signaling: phospho-Erk1/2 T202/Y204 (Cat.No.4370); phospho-Erk1Y204/Erk2 Y187 (mouse D1H6G); Erk1/2 (mouse 4696); phospho-MEK1/2 S217/221 (9154); MEK (mouse 4694); phospho-EGFR Y1173 (4407); EGFR (mouse 2239); Elk-1(rabbit Ab 9182), p-Elk1 (Ser383 mouse Ab 9186), MSK1 (rabbit Ab 3489), p-MSK1 (Thr 581 rabbit Ab 9595) and Erk Rabbit Ab 4695).
Active Ras assay was performed using the Active Ras Pull Down and Detection kit from Thermo Fisher (Cat.No.16117).

All the primary antibodies were polyclonal, raised in rabbits. The following antibodies were used: SOD2, CAT, CASP3, CASP7, CASP9, BCL2, BAX, Nf-kB, ALB, B-ACT, p38, Akt, PPARg, AMPK, and Erk 1/2 which were from Thermo Fisher Scientific, Waltham USA. GAPDH was from Cell Signaling Technology, Danvers, MA, USA. In the case of some cell signaling proteins, the phosphorylated forms were also measured: p-Akt, p-p38, p-PPARg, p-Erk 1/2, and p-AMPK (antibodies from Thermo Fisher Scientific, Waltham USA). The secondary antibody was goat anti-rabbit IgG, HRP conjugated (Thermo Fisher Scientific, Waltham, MA, USA). The labeling protocol is described in detail in Section 4.5.

In the other group, the similar contractile injury was induced in the rat model. The contralateral and ipsilateral horns from the lumbar region of the spinal cord were dissected from segments L4-L6 using laminectomy under the influence of isoflurane (Kent Scientific). The isolated dorsal horn was then homogenized in Laemmli buffer at freezing temperatures at pH of 7.5, containing 0.5% SDS (sodium dodecyl sulfate), 1% protease inhibitor, and 50 mM Tris-HCl (Sigma-Aldrich). Proteins were isolated on SDS-polyacrylamide gel electrophoresis which were then transferred to the polyvinylidene difluoride membrane. Incubation of the membranes was performed using primary antibodies pan-GluN2B (Sigma-Aldrich), p38 MAPK, ERK1/2, and JNK (Thermo Fischer) and control with GAPDH (glyceraldehyde 3-phosphate dehydrogenase, Abbexa) at 40°C in separate chambers. Now, the membranes were incubated with goat anti-rabbit immunoglobulin G (IgG, Bio-Rad Antibodies) at room temperature for 1 hour. Now, proteins were boosted by chemiluminescence and visualized for comparison and interpretation. Density of the bands was studied and compared to that of the loading control bands.

Cells were harvested and lysed in RIPA buffer (Thermo Fisher Scientific, UK) containing protease inhibitor and PhosSTOP tablets (Roche, UK) and sample protein concentration determined using the Pierce^® BCA Protein Assay Kit (Thermo scientific, UK). SDS-PAGE was then performed, and proteins (20-30 μg) were transferred onto nitrocellulose membranes. Membranes were blocked with TBS-T 5% non-fat milk prior to overnight incubation with for primary antibodies at 4°C (used at 1:5000 unless stated otherwise). The following antibodies were used: anti-Cytohesin2 (sc-374640; Santa Cruz Biotechnology, 1:1000), anti-ARF6 (ARF-06; Cytoskeleton, 1:500), anti-GAPDH (CST #2118, UK), anti-p38 (CST #9212, UK), anti-p38 phospho (CST #9211, UK), anti-STAT3 (CST #9139, UK) and anti-STAT3 phospho (CST #9145, UK), anti-ERK1/2 phospho (CST #9101, UK), anti-ERK1/2 (CST #4695, UK), anti-c-Jun phospho (CST #9261, UK, 1:1000) and anti-c-Jun (CST #9165, UK). Membranes were then incubated with anti-rabbit (CST #7074P2, UK) or anti-mouse (CST #7076s, UK) anti-IgHRP-conjugated secondary antibodies for 1h at room temperature and washed three times in TBS-T. Expression signal was detected by ECL Western Blotting Substrate (Thermo scientific, UK) and the protein bands were quantified using GelAnalyzer 2010a software with the relative integrated density values normalised to ERK1/2 and GAPDH expression values.

CRC cells were incubated for 4 h with PMPs as described above for the detection of CXCR4, MMP-2, and MMP-9 and lysed using RIPA buffer (Sigma Aldrich) or incubated for 10 min with PMPs for the detection of nonphosphorylated and phosphorylated ERK1/2 and p38MAPK and lysed with M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific). The lysates (25 µg of protein) were then separated on an SDS‒PAGE 10% polyacrylamide gel, transferred to a nitrocellulose membrane and incubated with rabbit anti-human antibodies against nonphosphorylated p38MAPK (Cell Signaling) and ERK1/2 (Thermo Fisher Scientific) and phosphorylated p38MAPK (Cell Signaling) or mouse anti-human antibodies against CXCR4 (Thermo Fisher Scientific), MMP-2, MMP-9 (Thermo Fisher Scientific) and phosphorylated ERK1/2 (Thermo Fisher Scientific), followed by incubation with secondary goat anti-mouse or goat-anti-rabbit IgG (Santa Cruz Biotechnology) conjugated with horseradish peroxidase (HRP) (Additional file 2: Supplementary Methods).