Anti phospho creb ser133

The mice were sacrificed under isoflurane anesthesia on day 20 after incision and/or alcohol treatment and the ipsilateral L4-L6 lumbar spinal cord tissues were harvested. Proteins from the lumbar spinal cord tissues were extracted as described previously.20 (link),24 (link) Nuclear fraction of the extracted protein was used for detecting CREB and its phosphorylation.24 (link) β-actin and histone H3 were used as loading controls for N-cadherin and CREB, respectively. Protein concentration was determined using the bicinchoninic acid method. The following affinity-purified antibodies were used: anti-N-cadherin (1:1000, Cat. # ab76057, Abcam, USA), anti-CREB (1:1000, Cat. # ab32515, Abcam, USA), anti-phospho-CREB-Ser133 (1:5000, Cat. # ab32096, Abcam, USA), anti-β-actin (1:200000, Cat. # A5316, Sigma, USA), and anti-histone H3 (1:3000, Cat. # 17168-1-AP, Proteintech, USA). The intensities of bands were quantified with densitometry using Image J software (NIH, USA). The intensity values of N-cadherin bands were normalized with β-actin and expressed as a ratio of N-cadherin/β-actin, and the intensity values of the phospho-CREB-Ser133 (p-CREB) were normalized with total CREB and expressed as a ratio of p-CREB/CREB. The specificity of anti-N-cadherin, anti-CREB anti-phospho-CREB-Ser133 antibodies has been validated previously.25 (link)–27 (link)