Anti bhlhe40 hpa028921

Immunohistochemistry was performed using the following antibodies: anti-BHLHE40 (HPA028921; Atlas Antibodies), anti-phospho-AMPKα (Thr172) (40H9; Cell Signaling Technology), anti-PPM1A (D18C10), or anti-PPM1F antibody (ab200394; Abcam). Briefly, a paraffin-embedded block was sliced into 5-μm-thick sections. The sections were deparaffinized and antigen was retrieved by Target Retrieval Solution (Agilent Dako; pH6 for BHLHE40 and pH9 for phospho-AMPKα, PPM1A, and PPM1F), then endogenous peroxidase was blocked with 0.3% hydrogen peroxide in methanol. The sections were incubated overnight with diluted primary antibody (1/200 in Antibody Diluent; Agilent Dako), then incubated with Envision+ Dual Link HRP (Agilent Dako) and visualized with 3, 3′ diaminobenzidine as a substrate. Hematoxylin was used for counterstaining. Specific staining was evaluated by the Allred scoring system (57 (link)).

Immunohistochemistry was performed as described previously using antibodies as follows: anti-BHLHE40 (HPA028921, Atlas Antibodies, Stockholm, Sweden) or anti-BHLHE41 (E-4, Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibody [8 (link)]. The nuclear expression of BHLHE40/41 was evaluated using a staining scoring system modified from that described by Allred et al. [48 (link)]. Staining scores were calculated by multiplying the proportion score by the intensity score.

Cell lysates for immunoblotting were prepared with cell lysis buffer (20 mM HEPES, pH 7.9, 0.5 N NaCl, 1 mM EDTA, 25% glycerol, 1% Ninodet P-40, 0.5 mM dithiothreitol, and 0.1% sodium deoxycholate) containing protein inhibitor and phosphatase inhibitor (Nacalai Tesque). After separation by electrophoresis, the proteins were transferred to the PVDF membrane (Immobilon, Merck Millipore). Immunoblotting was performed using the following primary antibodies: anti-BHLHE40 (HPA028921) from Atlas Antibodies, anti-AMPKα (D5A2), anti-phospho-AMPKα Thr172 (40H9), anti-AMPKβ (57C12), anti-phospho-AMPKβ1 Ser182 (4186), anti-ACC (C83B10), anti-phospho-ACC Ser79 (D7D11), anti-PPM1A (D18C10), anti-PDHA1 (C54G1), anti-phospho-PDHA1 Ser293 (31,866), anti-LDHA (C4B5), anti-phospho-LDHA Tyr10 (8167), anti-LKB1 (D60C5), anti-phospho-LKB1 Ser428 (C67A3), and anti-β-actin (13E5) (Cell Signaling Technology). Anti-PPM1B (ab70804), anti-PPM1E (ab137122), and anti-PPM1F (ab200394) antibodies were from Abcam. Anti-GAPDH (FL-335) antibody was from Santa Cruz Biotechnology. Anti-FLAG (M5) antibody was from Sigma–Aldrich (9 (link)). The intensity of blotting was semi-quantified using Image J software (https://imagej.net/ij/).