Ecl detection kit

After culturing for 3 h, the cells were lysed using NP40 buffer (ThermoFisher, MA, USA), and a BCA protein assay kit was used to obtain the total protein concentrations from the cell lysates. Next, the cell lysates of 35 μg protein/sample were segregated using sodium dodecyl sulfate-polyacrylamide-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a Poly (vinylidene fluoride) membrane (Milipore, Billerica, MA, USA), before further immunoblotting procedures. After which, the transferred proteins were fixated using 2% BSA in TBST for 1 h, before immunoblotting for 2 h with the following antibodies: Primary anti-integrin β1, β-actin (GeneTex, San Antonio, TX, USA), anti-focal adhesion kinase (FAK), and anti-phospho-FAK (p-FAK). Subsequently, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (ECL detection kit, Invitrogen, Carlsbad, CA, USA) for 1 h, to allow visualization of protein bands using chemiluminescence. The expression levels of the various proteins were normalized to β-actin, for further quantification analysis.

The protein samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Invitrogen). Membranes were then blocked for 1 h in 5% fat-free milk (Roth) in 20 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.8 (TBS-Tween), followed by incubation with primary antibodies against the His-Tag, the peroxisomal biogenesis proteins PEX3, PEX19 and PEX14, the peroxisomal matrix enzyme catalase, the mitochondrial matrix enzyme superoxide dismutase 2 (SOD2) and the cytosolic protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (S2 Table) for 1 h at room temperature. The membranes were washed and incubated with the secondary antibody for 1 h at room temperature. Detection was performed depending on the enzyme conjugated to the secondary antibody (S3 Table), either with the Immun-Star-AP detection kit (Bio-Rad Laboratories) for secondary antibodies conjugated to alkaline phosphatase or the ECL detection kit (Invitrogen) for secondary antibodies conjugated to horseradish peroxidase. Protein bands were detected by exposing the membranes to Kodak BioMax films. To assure that equal amounts of protein were present, all membranes were stained with Coommassie Brilliant Blue (CBB) (Simply Blue Stain, Invitrogen) according to the manufacturer´s protocol after the Western blot procedure was completed.

Western blot analysis was performed as described previously60 (link). Briefly, cells were harvested and lysed with the RIPA buffer (50 mM Tris-HCl, 1% NP-40, 0.35% sodium-deoxycholate, 150 mM NaCl, 1 mM EDTA, pH 7.4, 1 mM phenylmethylsulfonyl fluoride, 1 mM NaF, 1 mM Na₃VO₄ and 10 μg/ml each of aprotinin, leupetin and pepstatin A). Equal amount of protein samples were separated by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) and then electro-transferred onto nitrocellulose membranes (Amersham Biosciences, USA). After blocked with 5% milk in the TBST buffer for 1 h, membranes were subsequently probed with appropriate primary antibodies overnight at 4 °C. The membranes were then incubated with HRP-conjugated secondary antibodies. Immunoreactive bands were visualized using the ECL detection kit (Invitrogen, USA).

The quantified joint tissue lysates were assessed by Western blot analysis as described previously23 . Briefly, protein lysates were subjected to 10% SDS-PAGE and then electro-transferred onto nitrocellulose membrane. After blocking with 5% non-fat milk in TBST, the membrane was incubated with designated primary antibodies overnight. The membrane was then incubated with anti-mouse or anti-rabbit secondary antibodies48 (link)49 (link). The specific immunoreactive bands were detected using enhanced chemiluminescence ECL detection kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instruction.

After 3 h of culture, cells on scaffolds were lysed with NP40 buffer (ThermoFisher). The total protein concentrations were determined using BCA protein assay kit. The cell lysates (40 μg protein) were separated using sodium dodecyl sulfate-polyacrylamide (SDS)-polyacrylamide gel electrophoresis and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking with 2% BSA in TBST for 1 h, the membranes were immunoblotted with the primary anti-pFAK, anti-FAK, and β-actin (GeneTex, San Antonio, TX, USA) for 2 h. The bands were then visualized after incubation for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibodies by chemiluminescence using an ECL detection kit (Invitrogen, Carlsbad, CA, USA). The protein expression level was normalized to the β-actin for each group.

Immunoblotting of protein samples prepared from CRC cells or tumor tissues was performed following a previous study (Zhu et al., 2019 (link)). The protein samples were separated by 10–12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, United States). The membranes were then blocked with 5% (w/v) skimmed milk for 1 h and incubated with corresponding primary antibodies at 4°C overnight. Subsequently, the membranes were washed with Tris-buffered saline buffer with 0.1% Tween-20 (TBST) and incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. After washing with TBST, immunoreactive bands were visualized using enhanced chemiluminescence (ECL) detection kit (Invitrogen, Waltham, MA, United States) following the manufacturer’s instructions. Finally, the grey value of each band was measured using Image J software.

Cells were harvested and lysed in NP-40 lysis buffer (Fluka, USA) with protease and phosphatase inhibitors (ThermoFisher). Protein concentrations were measured using the Bradford Protein Assay Kit (Bio-Rad, USA). Equal amounts of protein were separated using 12% SDS-PAGE and transferred to PVDF membranes (Bio-Rad). Afterward, the membranes were blocked with 5% skim milk, incubated overnight at 4°C with each primary antibody, and then washed three times with TBST (25 mM Tris buffer, 0.15 M NaCl, 0.05% Tween 20). The membrane was incubated for 3 h at room temperature with the corresponding horseradish peroxidase (HRP)- conjugated secondary antibody and rewashed three times with TBST. Proteins were visualized using the ECL detection kit (Invitrogen). The antibodies used are as follows: Primary antibodies: rabbit anti-COX-2; rabbit anti- iNOS; rabbit anti-phospho and total-p44/p42 MAPK (Erk1/2); rabbit anti-phospho and total-p38 MAPK; rabbit anti-phospho and total JNK; rabbit anti-IkB (all antibodies mentioned above were from Cell Signaling Technology, USA). Secondary antibodies: mouse anti-β-actin (Sigma-Aldrich, USA); and HRP-conjugated secondary antibody (Promega).